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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

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Characterization and redesign of galactonolactone dehydrogenase, a flavoprotein producing vitamin C
Leferink, N.G.H. - \ 2009
Wageningen University. Promotor(en): Sacco de Vries, co-promotor(en): Willem van Berkel. - [S.l.] : S.n. - ISBN 9789085853527 - 168
oxidoreductasen - arabidopsis thaliana - enzymologie - oxidoreductases - enzymology
Keywords: aldonolactone oxidoreductases, Arabidopsis thaliana, flavoprotein, galactonolactone dehydrogenase, molecular gatekeeper, oxidase, protein engineering, vanillyl-alcohol oxidase family, vitamin C

Redox enzymes are attractive biocatalysts because of their intrinsic (enantio-)selectivity and catalytic efficiency, which are often difficult to achieve by conventional chemical approaches. The discovery of new redox enzymes together with novel insights into their catalytic mechanism will increase the biocatalytic potential for application of these enzymes. Carbohydrate oxidases are valuable enzymes that can be applied in diagnostics and the food industry. Such enzymes often contain a flavin cofactor as redox-active group. Most carbohydrate oxidase are up to now isolated from fungi, but an extensive genome analysis revealed that also plants are a rich source of these enzymes. Carbohydrate oxidases are, for example, involved in maintenance of the plant cell wall, de protection against pathogens, and the production of vitamin C.
In this research the vitamin C producing enzyme galactonolactone dehydrogenase from the model plant Arabidopsis thaliana was studied. This enzyme is a so-called aldonolactone oxidoreductases that belongs to the vanillyl-alcohol oxidase family of flavoproteins. Most members of this family are oxidases with a covalently bound FAD cofactor. Galactonolactone dehydrogenase differs in some crucial properties from aldonolactone oxidoreductases from animals, yeasts and fungi. The plant enzyme contains a non-covalently bound FAD cofactor, has a different substrate specificity and hardly reacts with molecular oxygen. Several critical amino acid residues involved in cofactor and substrate binding were identified and the enzyme was redesigned into variants with altered substrate and electron acceptor specificities. One of the major results was the identification of a gatekeeper residue in galactonolactone dehydrogenase that prevents molecular oxygen from reacting with the flavin cofactor. Removal of this residue resulted in a catalytically competent galactonolactone oxidase that can efficiently react with oxygen.
The knowledge obtained with this research provides a firm basis for the design of suitable biocatalysts that can be used for the biotechnological production of vitamin C or related carbohydrates, as an alternative for the currently applied chemical methods.

In vitro and in vivo interplay between NAD(P)H: quinone oxidoreductase 1 and flavonoids
Lee-Hilz, Y.Y. - \ 2007
Wageningen University. Promotor(en): Ivonne Rietjens; Sacco de Vries. - Wageningen : - ISBN 9789085047902 - 165
oxidoreductasen - flavonoïden - in vitro - in vivo experimenten - oxidoreductases - flavonoids - in vivo experimentation
Flavonoids are naturally occurring, health-promoting, bioactive compounds, omnipresent in the human diet. The protective effect of these phytochemicals is accomplished for an important part by modulating the activity of enzyme systems responsible for deactivation of chemical carcinogens, such as NAD(P)H: quinone oxidoreductase 1 (NQO1). Several flavonoids act as NQO1 inducers by increasing the NQO1 gene expression level through the electrophile-responsive element (EpRE). On the other hand certain flavonoids are efficient inhibitors of the NQO1 enzyme activity in vitro. The objective of this thesis is to elucidate the complex interplay between flavonoids and NQO1. First, inhibition of NQO1 by flavonoids, pointing at a mechanism contradicting the proven beneficial properties of these natural compounds was studied. Kinetic and molecular dynamics studies were conducted and a method to monitor NQO1 activity in living cells was developed. These studies revealed that although flavonoids possess the potential to inhibit NQO1 activity, inhibition of NQO1 is not likely to happen in cellular systems due to intracellular physiological conditions. Furthermore, the mechanism by which flavonoids are able to induce the EpRE- mediated expression of NQO1 was studied. Reporter gene assays elucidated that upstream XRE-mediated gene expression is not nessessary to induce EpRE-mediated gene expression and quantum-mechanical calculations revealed that flavonoids with a higher intrinsic potential to generate oxidative stress and redox cycling, are the most potent inducers of NQO1. Radioactive binding studies showed Keap1 modification by the flavonoid quercetin, resulting in switching on of EpRE-mediated gene transcription activation. In addition, in vivo metabolites of quercetin were studied on their ability to induce EpRE-mediated gene expression. The results show, that, although quercetin-derived glucuronides are the major metabolites present in the systemic circulation, the deglucuronidated parent compound and its methylated derivatives are the active compounds responsible for the beneficial EpRE-mediated gene expression effects. Overall, the studies presented in this thesis provide insight in the complex interplay between NQO1 and flavonoids on the protein as well as on the gene expression level.
Integrated molecular analysis of sugar metabolism of Sulfolobus solfataricus
Brouns, S.J.J. - \ 2007
Wageningen University. Promotor(en): Willem de Vos; John van der Oost. - [S.l.] : s.n. - ISBN 9789085047131 - 166
arabinose - oxidoreductasen - kristaleiwitten - thermofiele bacteriën - oxidoreductases - crystal proteins - thermophilic bacteria - cum laude
cum laude graduation (with distinction)
CO metabolism of carboxydothermus hydrogenoformans and archaeoglobus fulgidus
Henstra, A.M. - \ 2006
Wageningen University. Promotor(en): Fons Stams. - [S.l.] : S.n. - ISBN 9085044081 - 117
anaërobe afbraak - anaërobe micro-organismen - koolmonoxide - microbiële afbraak - oxidoreductasen - anaerobic digestion - anaerobes - carbon monoxide - microbial degradation - oxidoreductases
Hydrogenases and formate dehydrogenases of Syntrophobacter fumaroxidans
Bok, F.A.M. de; Roze, E.H.A. ; Stams, A.J.M. - \ 2002
Antonie van Leeuwenhoek: : Nederlandsch tijdschrift voor hygiëne, microbiologie en serologie 81 (2002)1-4. - ISSN 0003-6072 - p. 283 - 291.
micro-organismen - formiaten - hydrogenase - oxidoreductasen - microorganisms - formates - oxidoreductases
The syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans possesses two distinct formate dehydrogenases and at least three distinct hydrogenases. All of these reductases are either loosely membrane-associated or soluble proteins and at least one of the hydrogenases is located in the periplasm. These enzymes were expressed on all growth substrates tested, though the levels of each enzyme showed large variations. These findings suggest that both H2 and formate are involved in the central metabolism of the organism, and that both these compounds may serve as interspecies electron carriers during syntrophic growth on propionate.
Structural studies on metal-containing enzymes: T4 endonuclease VII and D. gigas formate dehydrogenase
Raaijmakers, H.C.A. - \ 2001
Wageningen University. Promotor(en): N.C.M. Laane; D. Suck. - S.l. : S.n. - ISBN 9789058084125 - 85
enzymen - röntgenkristallografie - desulfovibrio - oxidoreductasen - wolfraam - enzymes - x ray crystallography - oxidoreductases - tungsten
<p>Many biological processes require metal ions, and many of these metal-ion functions involve metalloproteins. The metal ions in metalloproteins are often critical to the protein's function, structure, or stability. This thesis focuses on two of these proteins, bacteriophage T4 endonuclease VII (EndoVII) and D. gigas fonnate dehydrogenase, which are studied by X-ray crystallography. The structure of EndoVII reveals how a magnesium or calcium ion is used to cleave several kinds of irregular but flexible DNA, while a zinc ion maintains the structural integrity of this DNase.</p><p>The formate dehydrogenase contains a tungsten ion and a seleno-cysteine at the active site, that catalyses the oxidation of formate to carbon dioxide. The two released electrons are transferred through four [4Fe-4S] clusters before they can be handed over to another protein. Two of the [4Fe-4S] and the selenium have been overlooked by other techniques, but could be localised and identified by crystallography.</p><p><strong>Chapter 1</strong> gives a general introduction on metals in biological systems, X-ray crystallography and also describes the biological background of both proteins.</p><p><strong>Chapter 2</strong> presents the structure of the four-way DNA-junction resolving enzyme T4 endonuclease VII, and that of the inactive N62D mutant. The betterexpressed mutant was solved first, using seleno-methionine, mercury and gold derivatives. These mercury and gold derivatives bind to the sulphurs that also ligand the zinc. The wild-type was solved with help of a single mercury derivative since molecular replacement with the mutant structure failed.</p><p>On its own, the EndoVII monomer would not represent a stable fold, as it exposes many hydrophobic residues to the solvent. But two monomers intertwine to form a dimer without this problem. In this dimer, the monomers are aligned head-to-tail; the N-terminus of one monomer interacts with the C-terminus of the other monomer and <em>vice versa</em> . The major dimerization element, unique to EndoVII, is the "four-helix-cross" domain, which consists of helix-2 and helix-3 from each monomer. It contains an extended hydrophobic core.</p><p>Another feature is the "beta-finger", residues 38-56. Its stability depends critically on the zinc. This zinc ion is tetrahedrally co-ordinated to four cysteines, linking helix- I through residues C23 and C26 firmly to the N-terminal part of helix-2 (C58, C61). Indeed, interfering mutations inactivate the protein. Finally, the calcium ion, which marks the active site, is liganded to aspartate-40 and asparagine-62. Mutation studies show that these amino acids are essential for activity: The N62D mutant is completely inactive.</p><p>The EndoVII structure has been docked to a "stacked-X" four-way DNA junction, one of its many substrates. This model is not refined, since both the DNA and the protein are known to be flexible and might undergo conformational changes. However, its overall features confirm experimental data: 1) The EndoVII dimer binds to the minor groove side of the four-way junction; 11) Basic residues on helix-2 can interact with phosphates on the exchanging strands and those on the C-terminal domain can interact with phosphates in the continuous strands, consistent with observed foot-printing patterns; 111) The C-terminus binds up to nine base pairs away from the junction, confirming the minimal length of two arms of the substrate; IV). The active sites do not cleave both the scissile phosphates simultaneously.</p><p>Surprisingly, the N62D mutant shows a major rearrangement in the "four-helixcross" domain, when compared to the wild-type: helices-2 are translated by half a turn each, in opposite direction and the opening of the "bays", between each helix-2 and betafinger, is wider. These differences might be attributed to the point-mutation, which introduces an extra charge in the active site, to differences in crystallisation conditions, to the different pH employed, to crystal contacts or perhaps they are simply a sign of the intrinsic flexibility of EndoVII.</p><p>This dilemma is partly solved in <strong>chapter 3, which</strong> presents the crystal structure of wild-type EndoVII in a different space group, which contains less solvent. It crystallised in the same drop, so that differences observed between the two wild-type structures cannot be attributed to the mutation, pH or salt concentrations. Since the helical-cross region of this second structure is very similar to that of the mutant, rearrangements in this region must be seen as a consequence of intrinsic flexibility of EndoVII. The widening of the "bays", however, might still be a consequence of the mutation, different pH, absence of Ca <sup>2+</sup> or crystal packing. An investigation of the flexibility of EndoVII with TLS- refinement, i.e. anisotropic refinement of rigid bodies, provides only limited insight. However, it confirms that rotations along the axes of the helices 2 and 4 and along the beta-finger are a main source of flexibility and also that the C-terminus, helix-4, 5 and 6, behave as a rigid body.</p><p>The high-resolution structure of the N62D mutant brings more clarity towards the reaction mechanism of the nuclease. This model contains important water molecules and reveals the position and orientation of 14 sulphate ions, which may indicate favoured phosphate (DNA) binding sites. Supported by new mutation data (Birkenbihl, unpublished), these sulphate and water positions, combined with the Ca <sup>2+</sup> positions in the wild-type structures, suggest a reaction-mechanism similar to those proposed for some other magnesium dependent nucleases.</p><p>2 Asparagine-62, glutamate-65 and aspartate-40 are important to position Mg <sup>2+</sup> or Ca <sup>2+</sup> next to the scissile phosphate of the DNA substrate. Histidine-41 activates a water molecule, which in turn executes a nucleophilic attack on the phosphor atom. Histidine-43 stabilises this phosphate directly through a hydrogen bond. Unfortunately it is still unclear why the N62D shows no DNase activity at all; an aspartate would also be able to ligand/position a divalent cation. The extra charge that this mutation introduces in the active site might distort the geometry of the active site, and repel the DNA. A more attractive, albeit more speculative hypothesis, assumes that the amino group of asparagine-62 donates a hydrogen-bond to the phosphate, which would also stabilise the transition state.</p><p>At present, there are no known proteins with significant sequence homology to EndoVII, though nucleases with structural similarities do exist. One group consists of magnesium-dependent nucleases, which have a similar geometry of liganding sidechains around the magnesium (or calcium) ion in the active site; e.g. the E. coli proteins RuvC (Ariyosi et al., 1994) and RNase H (Katayanagi et al., 1990). However, these nucleases have no resembling fold. Most likely, this just shows that magnesium-dependent nucleases need a certain geometry to function.</p><p>A more interesting group shares a folding motif similar to the beta finger and helix-2: Serratia Nuclease (Miller et al., 1994), Ppol (Flick et al., 1998) and perhaps even Colicin E9 (Kleanthous et al., 1999). Asparagine-62 and histidine-41 are conserved between Serratia nuclease, Ppol and T4 endonuclease VII. Ppol has also been crystallised in complex with DNA. If one superimposes this with the EndoVII structure, it turns out that the Ca <sup>2+</sup> in EndoVII is buried deeper within the protein, but small rotations (10-20 degrees) along helix 2 and the beta-finger suffice to superimpose them. These two nucleases act on different substrates, and maybe the larger DNA junctions of EndoVII need a wider and deeper binding groove than the double stranded DNA of Ppol. However, it could also be the source of EndoVII's specificity; flexible DNA might impose this conformational change of EndoVII upon binding, readying the enzyme for cleavage, while the magnesium or calcium ion might be too far away if EndoVII approaches more rigid DNA. A structure of EndoVII in complex with DNA would solve these questions.</p><p><strong>Chapter 4</strong> presents the major part of the determination of the 3D structure of the tungsten-containing formate dehydrogenasc (W-FDH) from Desulfovibrio gigas, one of the first tungsten-containing enzymes isolated from a mesophile. The large subunit (92 kDa) is structurally related to several tungsten- and molybdenumcontaining enzymes and X-ray structures have been determined for two of them. One of these, the periplasmic nitrate reductase (Dias et al, 1999), could be used to obtain a molecular replacement solution. But the quality of phasing was not sufficient to generate a clear, interpretable electron density map. Furthermore, the amino acid sequence of W-FDH has not yet been determined, what makes model building complicated. Multiple wavelength diffraction (MAD) measurements were undertaken at the absorption edges of W and Fe to define unambiguously the number, positions and identity of these anomalous scatterers and to improve the X-ray phases. The MAD-analysis revealed one W-atom with a Se-cys ligand and one [4Fe-4S] cluster bound to the large subunit, and three [4Fe-4S] clusters in the small subunit. The four [4Fe-4S] clusters are ca. 10 Å apart, creating a feasible electron transfer pathway, which connects the exterior of the protein to the W/Se site in the large subunit. Two of the four iron-sulphur clusters had not been predicted before by spectroscopic techniques (Almendra et al., 1999). A reinvestigation of the spectroscopic data was performed, but gave the same results as before. If these data were correct, this means that the [4Fe-4S] clusters are instable, and that only protein with fully occupied clusters crystallises.</p><p>The formate dehydrogenase H (FDH-H) from E. coli catalyses the same reaction as W-FDH, but uses a molybdenum instead of tungsten. Both are liganded to two molybdopterin-cofactors and to a seleno-cysteine, so the question remains why W-FDH prefers tungsten to the more common molybdenum. The full structure will allow a comparison of the two enzymes in atomic detail, and perhaps, it will shed some light on this phenomenon.</p><p>X-ray crystallography has been used to characterise the nature of metal-centres in proteins, their coordination geometry and even their identity. Sometimes, the way metal ions are bound to the protein already clarifies its role in the protein. In other cases it has to be supplemented with other studies before the role can be fully understood. Either way, crystallography provides a powerful tool for the study of metalloproteins.</p><dl><dt><em>References</em></dt><dd>Almendra, M.J., Brondino, C.D., Gavel, 0., Pereira, A.S., Tavares, P., Bursakov, S., Duarte, R., Caldeira, J., Moura, J.J.G., Moura, 1. (1999) <em>Biochemistry, 38</em> , 16366-16372</dd><dd>Ariyosi, M., Vassylyev, D., Iwasaki, H., Shinagawa, H. and Morikawa, K. (1994) <em>Cell, 78</em> , 1063-1072.</dd><dd>Flick, K.E., Jurica, M.S., Monnart Jr, R.J. and Stoddard, B.L. (1998), <em>Nature, 394</em> , 96-101.</dd><dd>Katayanagi, M., Miyagawa, M., Matsushima, M., Ishikawa, M., Kanaya, S., Ikehara, M., Matsuzaki, M. and Morikawa, K. (1990) <em>Nature, 347</em> , 306-309.</dd><dd>Kleanthous, C., Kuhlmann, U.C., Pornmer, A.J., Ferguson, N., Radford, S.E., Moore, G.R., James, R. and Hemmings, A.M. (1999) <em>Nature Struct. Biol., 6</em> , 243-252.</dd><dd>Miller, M.D., Tanner, J., Alpaugh, M., Benedik, M.J. and Krause, K.L. (1994) <em>Nature Struct Biol., 1</em> , 461-468.</dd></dl>
Towards the integration of oxidative and reductive activities: application to nitrogen removal by co-immobilized microorganisms
Martins dos Santos, V.A.P. - \ 2001
Wageningen University. Promotor(en): J. Tramper; Rene Wijffels. - S.l. : S.n. - ISBN 9789058083906 - 353
immobilisatie - micro-organismen - stikstofretentie - oxidoreductasen - biodegradatie - nitrificatie - immobilization - microorganisms - nitrogen retention - oxidoreductases - biodegradation - nitrification
<h3>Background</h3><p>Complete degradation of many pollutants requires sequenced anaerobic-aerobic biotreatment steps. Many compounds that are difficult to degrade aerobically are readily biotransformed anaerobically. The products of anaerobic biotransformation, however, will frequently resist to further mineralization; yet, they will be good substrates for aerobic biodegradation. Examples of this are the sequential biodegradation of highly chlorinated aromatics and aliphatics, azo-dyes, TNT, inorganic nitrogen compounds (NH <sub>4</sub><sup>+</sup> , NO <sub>2</sub><sup>-</sup> and NO <sub>3</sub><sup>-</sup> ) and pesticides such as DDT, HCH's or methoxychlor. In waste-and groundwater treatment, these sequenced biotransformations are commonly achieved either by using aerobic and anaerobic (anoxic) reactors in series or by alternating periods of aerobiosis and anaerobiosis in a treatment unit. Ideally, however, these biodegradation processes would take place in a single, compact continuous-reactor system under carefully controlled conditions. In many instances, the benefits of such an integrated system would be clearly greater than the mere sum of the advantages of each individual process.</p><h3>Magic beads: an advanced engineering concept for process integration</h3><p>This work addresses the possibilities of integrating oxidative and reductive complementary biodegradation processes in compact systems by using co-immobilized mixed-culture systems. The central idea throughout the book is that aerobic and anaerobic niches will eventually develop and coexist within a single biocatalytic particle so that oxidative and reductive activities (e.g nitrification and denitrification, respectively) can be accomplished simultaneously (Magic-beads). Therefore, multiple-step complementary biodegradation and biotransformation processes could be conducted as single staged (Figure 1). The rationale behind this idea relies on sound experimental evidence (e.g. time-dependent measurements of oxygen gradients across biocatalyst particles or biofilms) that shows that such niches do indeed establish under aerobic process conditions.</p><div align="center"><img src="/wda/abstracts/i2958_1.gif" width="492" height="307" border="0" alt="Figure 1 - Schematic representation of the "Magic-bead Concept"."/><br/>Figure 1 - Schematic representation of the "Magic-bead Concept".</div><h3>Case study: Integrated nitrification and denitrification</h3><p>The potential of the general concept outlined above for combining oxidative and reductive processes with relevance to the biodegadation of recalcitrant compounds is assessed in this work by studying in detail coupled nitrification and denitrification within (double-layered) gel beads for high-rate removal of nitrogen from wastewaters. In such beads, the nitrifying microorganisms (aerobes) immobilized in an outer layer would oxidize ammonium into nitrite that would then diffuse inwards, where immobilized denitrifiers (either facultative heterotrophs or obligate anaerobic ammonium oxidizers) would reduce this nitrite into the harmless gaseous nitrogen. The biocatalyst particle is used optimally because both the external layers and core are active. The beads are placed in a common airlift reactor through which the waste streams can flow at almost any rate, without the need of recirculation to or from any anoxic compartment or reactor.</p><h3>Aims of the dissertation and outline</h3><p>This research project aimed at a) the development and characterization of a coupled system for integrated nitrogen removal, b) understanding the mechanisms underlying the processes involved and; c) providing knowledge for the integration of oxidative and reductive activities in a single compact system. With these aims in mind, the stepwise strategy depicted in Figure 2 was developed. Every stage of the project was comprised by a series of self-contained studies addressing different aspects of the proposed system.</p><div align="center"><img src="/wda/abstracts/i2958_2.gif" width="550" height="500" border="0" alt="Figure 2 - Structured contents of this dissertation."/><br/>Figure 2 - Structured contents of this dissertation.</div><p>In the first stage (chapter 2) the scope of the problem was defined (apparent incompatibility of oxidative and reductive activities of environmental relevance), the needs were addressed (urge to integrate processes and reduce reactor size) and the state-of- the-art of the field (conventional systems and emerging technologies) was presented. In the following phase (chapters 3 to 6), a compact process was proposed and the procedures for biocatalyst production, and its characterization and mechanical stability were assessed. In the next stage (chapter 7 to 9), achievement of in-depth insight into the system's behavior was pursued by means of mathematical modeling and concomitant experimental validation using specific microelectrodes. The knowledge gathered up to this point was subsequently used successfully for the design of a fully autotrophic system for nitrogen removal (chapters 10 and 11). Finally, the possibilities of integrating other oxidative and reductive complementary biodegradation processes in compact systems by using co-immobilized mixed-culture systems were discussed in Chapter 12.</p>
Function, mechanism and structure of vanillyl-alcohol oxidase
Fraaije, M.W. - \ 1998
Agricultural University. Promotor(en): N.C.M. Laane; Willem van Berkel. - S.l. : Fraaije - ISBN 9789054858287 - 182
oxidoreductasen - chemische structuur - moleculaire structuur - reactiemechanisme - oxidoreductases - chemical structure - molecular conformation - reaction mechanism
<p>Lignin is a heterogeneous aromatic polymer formed by all higher plants. As the biopolymer lignin is a major constituent of wood, it is highly abundant. Lignin biodegradation, an essential process to complete the Earth's carbon cycle, is initiated by action of several oxidoreductases excreted by white-rot fungi. The resulting degradation products may subsequently be used by other microorganisms. The non-lignolytic fungus <em>Penicillium simplicissimum</em> can grow on various lignin metabolites. When this ascomycete is grown on veratryl alcohol, a major lignin metabolite, production of an intracellular aryl alcohol oxidase is induced. Purification and initial characterization revealed that this enzyme is able to oxidize vanillyl alcohol into vanillin and was therefore named: vanillyl-alcohol oxidase (VAO). Furthermore, it was found that VAO is a homooctamer of about 500 kDa with each subunit containing a covalently bound 8</font><FONT FACE="Symbol" SIZE=2>a</font><FONT FACE="Times,Times New Roman">-( <em>N</em></font><FONT FACE="Times,Times New Roman" SIZE=2>3</font><FONT FACE="Times,Times New Roman">-histidyl)-FAD redox group. As VAO showed some interesting catalytical and structural features, a PhD-project was started in 1993 with the aim of elucidating its reaction mechanism.</p><strong><p> </p></strong><p>In the initial stage this PhD-project, it was found that VAO has a rather broad substrate specificity. However, it was unclear which substrates are of physiological relevance. In a recent study, evidence was obtained that 4-(methoxymethyl)phenol represents a physiological substrate (Chapter 2). When the fungus is grown on 4-(methoxymethyl)phenol, VAO is expressed in large amounts, while the phenolic compound is fully degraded. HPLC analysis showed that VAO catalyzes the first step in the degradation pathway of 4-(methoxymethyl)phenol (Fig. 1).</p><p><img src="/wda/abstracts/i2416_1.gif" width="480" height="113"/></p><p>Figure 1.</strong> Degradation pathway of 4-methoxymethyl)phenol in <em>Penicillium simplicissimum.</em></p><p>This type of reaction (breakage of an ether bond) is new for flavoprotein oxidases. Furthermore, 4-(methoxymethyl)phenol has never been described in the literature as being present in nature. Yet, it can be envisaged that this phenolic compound is formed transiently during the biodegradation of lignin, a biopolymer of phenolic moieties with many ether bonds.</p><p>Concomitant with the induction of VAO a relatively high level of catalase activity was observed. A further investigation revealed that <em>P. simplicissimum</em> contains at least two hydroperoxidases both exhibiting catalase activities: an atypical catalase and a catalase-peroxidase (Chapter 3). Purification of both enzymes showed that the periplasmic atypical catalase contains an uncommon chlorin-type heme as cofactor. The intracellular catalase-peroxidase represents the first purified dimeric eucaryotic catalase-peroxidase. So far, similar catalase-peroxidases have only been identified in bacteria. These procaryotic hydroperoxidases show some sequence homology with cytochrome <em>c</em> peroxidase from yeast which is in line with their peroxidase activity. EPR experiments revealed that the catalase-peroxidase from <em>P. simplicissimum</em> contains a histidine as proximal heme ligand and thereby can be regarded as a peroxidase-type enzyme resembling the characterized procaryotic catalase-peroxidases.</p><p>In Chapter 4, the subcellular localization of both VAO and catalase-peroxidase in <em>P. simplicissimum</em> was studied by immunocytochemical techniques. It was found that VAO and catalase-peroxidase are only partially compartmentalized. For both enzymes, most of the label was found in the cytosol and nuclei, while also some label was observed in the peroxisomes. The similar subcellular distribution of both oxidative enzymes suggests that catalase-peroxidase is involved in the removal of hydrogen peroxide formed by VAO. The VAO amino acid sequence revealed no clear peroxisomal targeting signal (PTS). However, the C-terminus consists of a tryptophan-lysine-leucine (WKL) sequence which resembles the well-known PTS1 which is characterized by a C-terminal serine-lysine-leucine (SKL) consensus sequence.</p></strong><p>Soon after the start of the project, it was discovered that, aside from aromatic alcohols, VAO also converts a wide range of other phenolic compounds, including aromatic amines, alkylphenols, allylphenols and aromatic methylethers (Chapter 5). Based on the substrate specificity (Fig. 2) and results from binding studies, it was suggested that VAO preferentially binds the phenolate form of the substrate. From this and the relatively high pH optimum for turnover, it was proposed that the vanillyl-alcohol oxidase catalyzed conversion of 4-allylphenols proceeds through a hydride transfer mechanism involving the formation of a <em>p</em> -quinone methide intermediate.</p><p><img src="/wda/abstracts/i2416_2.gif" width="512" height="498"/></p><p>Figure 2. Reactions catalyzed by VAO.</p><p>In Chapter 6, the kinetic mechanism of the oxidative demethylation of 4-(methoxymethyl)phenol was studied in further detail using the stopped-flow technique. It was established that the rate-limiting step during catalysis is the reduction of the flavin cofactor by the aromatic substrate (Fig. 3). Furthermore, it was found that during this step a binary complex is formed between the reduced enzyme and a product intermediate. Spectral analysis revealed that the enzyme-bound intermediate is the <em>p</em> -quinone methide form of 4-(methoxymethyl)phenol. Upon reaction of this complex with molecular oxygen, the final product is formed and released in a relatively fast process. Using H</font><FONT FACE="Times,Times New Roman" SIZE=2>218</font><FONT FACE="Times,Times New Roman">O, we could demonstrate that, upon flavin reoxidation, water attacks the electrophilic quinone methide intermediate to form the aromatic product 4-hydroxybenzaldehyde.</p><p><img src="/wda/abstracts/i2416_3.gif"/></p><p>Figure 3.</strong> Reaction mechanism for the oxidative demethylation of 4-(methoxymethyl)phenol.</p><p>In Chapter 7, the enantioselectivity of VAO was investigated. VAO catalyzes the enantioselective hydroxylation of 4-ethylphenol, 4-propylphenol and 2-methoxy-4-propylphenol with an <em>ee</em> of 94% for the R-enantiomer. Isotope labeling experiments confirmed that the oxygen atom incorporated into the alcoholic products is derived from water. During the VAO-mediated conversion of short-chain 4-alkylphenols, 4-alkenylic phenols are produced as well. The reaction of VAO with 4-alkylphenols also results in minor amounts of phenolic ketones which is indicative for a consecutive oxidation step.</p><p>Also the kinetic mechanism of VAO with 4-alkylphenols was studied (Chapter 8). For the determination of kinetic isotope effects, C</font><FONT FACE="Symbol" SIZE=2>a</font><FONT FACE="Times,Times New Roman">-deuterated analogues were synthezised. Interestingly, conversion of 4-methylphenol appeared to be extremely slow, whereas 4-ethyl- and 4-propylphenol were rapidly converted. With these latter two substrates, relatively large kinetic deuterium isotope effects on the turnover rates were observed indicating that the rate of flavin reduction is rate-limiting. With all three 4-alkylphenols, the process of flavin reduction was reversible with the rate of reduction being in the same range as the rate of the reverse reaction. With 4-ethylphenol and 4-propylphenol, a transient intermediate is formed during the reductive half-reaction. From this and based on the studies with 4-(methoxymethyl)phenol, a kinetic mechanism was proposed which obeys an ordered sequential binding mechanism. With 4-ethylphenol and 4-propylphenol, the rate of flavin reduction determines the turnover rate, while with 4-methylphenol, a step involved in the reoxidation of the flavin seems to be rate limiting. The latter step might be involved in the decomposition of a flavin N5 adduct.</p><p>During crystallization experiments it was found that VAO crystals are highly sensitive towards mercury and other heavy atom derivatives. Therefore, the reactivity of VAO towards mercury in solution was studied (Chapter 9). Treatment of VAO with <em>p</em> -mercuribenzoate showed that one cysteine residue reacts rapidly without loss of enzyme activity. Subsequently, three sulfhydryl groups react leading to enzyme inactivation and dissociation of the octamer into dimers. From this, it was proposed that subunit dissociation accounts for the observed sensitivity of VAO crystals towards mercury compounds.</p></strong><p>Recently, the crystal structure of VAO was solved (Chapter 10). The VAO structure represents the first crystal structure of a flavoenzyme with a histidyl bound FAD. The VAO monomer comprises two domains (Fig. 4).</p><p><img src="/wda/abstracts/i2416_4.gif"/></p><strong><p>Figure 4.</strong> Crystal structure of VAO at 0.25 nm resolution.</p><p>The larger domain forms a FAD-binding module while the other domain, the cap domain, covers the reactive part of the FAD cofactor. By solving the binding mode of several inhibitors, the active site of VAO could be defined. This has clarified several aspects of the catalytic mechanism of this novel flavoprotein. Three residues, Tyr</font><FONT FACE="Times,Times New Roman" SIZE=2>108</font><FONT FACE="Times,Times New Roman">, Tyr</font><FONT FACE="Times,Times New Roman" SIZE=2>503</font><FONT FACE="Times,Times New Roman">and Arg</font><FONT FACE="Times,Times New Roman" SIZE=2>504</font><FONT FACE="Times,Times New Roman">, are involved in substrate activation by stabilizing the phenolate form of the substrate. This is in line with the proposed formation and stabilisation of the <em>p</em> -quinone methide intermediate and the substrate specificity of VAO. The structure of the enzyme 4-heptenylphenol complex revealed that the shape of the active-site cavity controls substrate specificity by providing a 'size exclusion mechanism'. Furthermore, the active site cavity has a rigid architecture and is solvent-inaccessible. A major role in FAD binding is played by residues 99-110, which form the so-called 'PP loop'. This loop contributes to the binding of the adenine portion of FAD and compensates for the negative charge of the pyrophosphate moiety of the cofactor. The crystal structure also established that the C8-methyl group of the isoalloxazine ring is linked to the N</font><FONT FACE="Symbol" SIZE=2>e</font><FONT FACE="Times,Times New Roman">2 atom of His</font><FONT FACE="Times,Times New Roman" SIZE=2>422</font><FONT FACE="Times,Times New Roman">. Intriguingly, this residue is located in the cap domain.</p></strong><p>From the crystallographic data and sequence alignments, we have found that VAO belongs to a new family of structurally related flavin-dependent oxidoreductases (Chapter 11). In this study, 43 sequences were found, which show moderate homology with the VAO sequence. As sequence homology was mainly found in the C-terminal and N-terminal parts of the proteins, it could be concluded that the homology is indicative for the conservation of a novel FAD-binding domain as was found in the crystal structure of VAO (Fig. 5). This structurally related protein family includes flavin-dependent oxidoreductases isolated from (archae)bacteria, fungi, plants, animals and humans, indicating that this family is widespread. Furthermore, the sequence analysis predicts that many members of this family are covalent flavoproteins containing a histidyl bound FAD.</p><p><img src="/wda/abstracts/i2416_5.gif"/></p><p>Figure 5.</strong> Schematic drawing of the structural fold of the newly discovered flavoprotein family.</p></strong><p></p><p>Some of the VAO-mediated reactions are of relevance for the flavour and fragrance industry. For example, reactions of VAO with vanillyl alcohol, vanillylamine or creosol all result in the formation of vanillin, the major constituent of the well-known vanilla flavour. Furthermore, as shown in Chapter 7, VAO is able to enantioselectively hydroxylate phenolic compounds resulting in the production of interesting synthons for the fine-chemical industry. Because of its versatile catalytic potential and as VAO does not need external cofactors, but only uses molecular oxygen as a cheap and mild oxidant, VAO may develop as a valuable tool for the biotechnological industry. Furthermore, the recent cloning of the VAO gene and the available crystal structure will allow protein engineering to redesign the catalytic performance of VAO, which is of main interest for biotechnological applications. Therefore, like glucose oxidase and</font><FONT FACE="Times,Times New Roman" SIZE=2>D</font><FONT FACE="Times,Times New Roman">-amino acid oxidase, VAO can be placed among an emerging group of flavoprotein oxidases, that catalyze transformations of industrial relevance.</p></font>
The glucose-6-phosphate dehydrogenase encoding genes from Aspergillus niger and Aspergillus nidulans
Broek, P. van den - \ 1997
Agricultural University. Promotor(en): C. Heyting; H.W.J. van den Broek. - S.l. : Van den Broek - 128
oxidoreductasen - oxidoreductases
<br/>Glucose-6-phosphate (G6P) is a central metabolite, that can either be metabolised via the glycolytic and tricarboxylic acid cycle to generate ATP, or converted into storage molecules or can be directed to the pentose phosphate pathway to yield NADPH and various pentoses. This thesis focuses on one of these G6P consuming reactions, catalysed by glucose-6-phosphate dehydrogenase (G6PD), in which G6P is oxidised and NADP <sup>+</SUP>acts as electron acceptor. The central theme of this thesis is the role of glucose-6- phosphate dehydrogenase (G6PD) enzyme activity in the generation of the cytosolic NADPH pool. This cytosolic NADPH pool serves as the reducing agent in a large number of biosynthetic reactions, in the protection against oxidation damage and in the utilisation of nitrate and certain pentoses. Only a limited number of catabolic reactions generate NADPH of which the one catalysed by G6PD is usually considered to be the most important.<p>If G6PD were the major producer of NADPH, one would expect 1) G6PD enzyme activity to respond to changes in NADPH demand and 2) G6PD mutations to be extremely deleterious or even lethal. According to these criteria, examples can be found in the scientific literature, where G6PD appears to be either vital for cytosolic NADPH production or appears to be completely superfluous. <em>Aspergillus nidulans</em> and <em>Aspergillus niger</em> were chosen as model systems because of their ability to metabolise a great variety of carbon- and nitrogen sources and their well developed genetics. These characteristics would allow us to use nitrogen- and carbon sources that influence NADPH consumption and to employ molecular biological techniques to study and manipulate the regulation of G6PD expression.<p>The biochemical characteristics of the purified G6PD enzymes from A. nidulans and <em>A. niger</em> suggest the involvement of these G6PD enzymes in NADPH production. For both pure enzyme preparations, the main modulator of G6PD enzyme activity is the redox potential as is reflected by its stimulation by NADP <sup>+</SUP>and its competitive inhibition by NADPH. Additionally, these observations show that at least part of the regulation of G6PD activity in <em>A. nidulans</em> and <em>A. niger</em> is exerted at the enzyme level. In their biochemical characteristics, their primary and secondary structure the G6PDs from A. nidulans and <em>A. niger</em> are virtually identical. Both fungal G6PD enzymes bind their substrates G6P and NADP <sup>+</SUP>in a random order and exhibit a strict specificity towards them. Whether the presence of two proteins in both pure Aspergillus G6PD preparations, has any physiological significance or is just an artefact due to proteolytic degradation of the native G6PD during the purification procedure, remains to be determined. However, the cloning of the G6PD encoding genes does provide a clue to the origin of the two G6PD proteins. In <em>A. nidulans</em> both G6PD proteins should be derived from the <em>gsd</em> A gene, since only a single G6PD encoding gene could be detected on genomic Southern blots. For the two <em>A. niger</em> G6PD proteins, the situation is less clear because genomic Southern blots revealed the presence of additional DNA bands that cross-hybridised strongly to the <em>gsd</em> A gene and which could represent a second G6PD encoding gene.<p>The <em>gsd</em> A genes from <em>A. nidulans</em> and <em>A. niger</em> have an identical structure; both contain nine introns, which are located at exactly equivalent positions with respect to the coding region. Furthermore, both genes exhibit strong DNA sequence homology in the coding region, but in the introns, 5'- and 3'-flanking sequences, this homology drops significantly. Alignment of the deduced <em>gsd</em> A amino acid sequences with other eukaryotic G6PDs reveals strong homology at the amino acid level and allows localisation of important domains like the putative catalytic site and NADP <sup>+</SUP>bindings sites. The cloning of the <em>gsd</em> A genes a] lows us to manipulate the G6PD activity directly. The increase of G6PD activity by the introduction of multiple copies of the <em>gsd</em> A gene results in <em>A. niger</em> in grossly disturbed growth especially on media containing reduced nitrogen sources. The degree of growth inhibition and the increase in G6PD activity are directly related to the number of functionally integrated <em>gsd</em> A genes. This observation suggests the reduced growth is a direct consequence of the increased G6PD activity. One can explain these phenomena by assuming that increased G6PD activity results in overproduction of NADPH. If for this excess of NADPH no acceptor is available (e.g. nitrate), the redox potential is disturbed and growth is inhibited. A similar experiment with the <em>A. nidulans</em> gene resulted in cotransformants with a single additional <em>gsd</em> A copy, that only slightly overexpressed G6PD end did not exhibit reduced growth.<p>This already shows that despite the similarity in the biochemical characteristics and in primary structure of the enzymes and genes, there are remarkable differences in the regulation of the <em>A. niger</em> and <em>A. nidulans</em><em>gsd</em> A expression. In <em>A. nidulans</em> , but not in <em>A. niger</em> , G6PD enzyme activity and <em>gsd</em> A transcription respond to the increased NADPH demand during growth on nitrate. These observations indicate that in <em>A. nidulans</em> G6PD does play an important role in NADPH production, whereas in <em>A. niger</em> it probably does not. Further evidence for this statement comes from the observation that in <em>A. niger</em> G6PD enzyme activity and steady state mRNA levels do not change in response to growth on xylose or in response to oxidation stress.<p>The homology in the <em>gsd</em> A 5'-upstream regions is limited to four 11 to 22 bp long sequence blocks, named <em>gsd</em> A-boxes, which in both promoters appear in the same order. Homologues of <em>gsd</em> A-boxes 1, 2 and 4 are also encountered in the same order in the <em>S. cerevisiae zwf</em> l promoter. In both Aspergilli, the <em>gsd</em> A-box region contains all transcription start sites: in the case of <em>A. niger</em> there are four and in <em>A. nidulans</em> there are as much as eight. We did not find any conservation, neither in the number of transcription sites nor the site of initiation. In <em>A. niger</em> all deletions within the region containing the <em>gsd</em> A- boxes, reduce transcription to background level while such a deletion in <em>A. nidulans</em> retains 25% of its original transcription. These observations indicate that despite the <em>gsd</em> A-box homology, these regions still differ functionally.<p>From the deletion study of the <em>A. nidulans</em><em>gsd</em> A promoter, it is clear that the nitrate induced increase in G6PD activity is caused by an increased <em>gsd</em> A transcription. Furthermore, evidence has been obtained that the NIRA transcription factor, which mediates induction of the nitrate utilisation pathway, is also involved in the nitrate stimulation of <em>gsd</em> A transcription in <em>A. nidulans</em> . Furthermore our data show clearly, that NIRA plays an important role in the regulation of <em>gsd</em> A transcription in the absence of nitrate. This phenomenon is probably yet another difference in the regulation of <em>gsd</em> A expression between <em>A. nidulans</em> and <em>A. niger</em> . Since <em>A. niger</em><em>gsd</em> A expression does not respond to nitrate induction, it is unlikely that the uninduced level of its <em>gsd</em> A transcription is under the control of NIRA. This NIRA dependence of <em>gsd</em> A transcription might also explain the remarkably different behaviour of the two <em>gsd</em> A genes in cotransformation experiments.<p>In <em>A. nidulans</em> , titration of the NIRA protein by the cotransformed <em>gsd</em> A gene might limit the expression of the cotransformed <em>gsd</em> A copies. At the same time cotransformants with multiple copies of <em>gsd</em> A would be lost on the nitrate containing selection medium, since NIRA titration would render them incapable of nitrate utilisation. These two phenomena would explain the low cotransformation frequency of the <em>A. nidulans</em><em>gsd</em> A gene and low G6PD overproduction observed in <em>A. nidulans</em> cotransformants. Conversely, the absence of NIRA regulation in the <em>gsd</em> A expression in <em>A. niger</em> does not limit overproduction of G6PD in <em>gsd</em> A cotransformants. Furthermore, the absence of NIRA titration in A. <em>niger g</em> sdA cotransformants allows the use of nitrate as an acceptor for the excess NADPH.<br/>In contrast to the other members of the nitrate utilisation regulon (e.g. <em>nia</em> D, <em>nii</em> A and <em>crn</em> A) is <em>gsd</em> A expression not subject to nitrogen catabolite repression exerted by the areA gene product. Still, AREA seems to act on the <em>A. nidulans</em><em>gsd</em> A promoter. The physiological significance of this phenomenon remains to be established.<p>The data presented in this thesis show that although the primary structure and biochemical characteristics of an enzyme from two different organisms are virtually identical, there can be significant differences in the regulation of their expression and hence in their physiological function. Of course our data provide only an outline of the regulation of G6PD activity in two Aspergilli and, unfortunately, raise more questions then they answer.<p>Our data indicate that G6PD does not play a crucial role in the generation of the cytoplasmic NADPH pool in <em>A. niger</em> . What is then the physiological function of G6PD enzyme activity in A. <em>niger?</em> Furthermore, as it is difficult to assign a physiological function to the <em>A. niger</em><em>gsd</em> A gene, is it reasonable to assume that the fragments that cross-hybridise to the <em>gsd</em> A gene in digests of <em>A. niger</em> genomic DNA could represent a second G6PD encoding gene? Clearly, the construction and characterisation of a <em>gsd</em> A null mutant should provide important clues to these questions. Since G6PD is not the main producer of NADPH in <em>A. niger</em> , a <em>gsd</em> A null mutation should be viable. This of course raises the question which pathway provides the bulk of the cytoplasmic NADPH in <em>A. niger</em> ?<p>In <em>A. nidulans</em> G6PD enzyme activity is clearly involved in the maintenance of a proper cytoplasmic NADP <sup>+</SUP>/NADPH ratio as its expression responds to the increased NADPH consumption during growth on nitrate. From this observation we would predict that a <em>gsd</em> A null mutant, if not lethal, would be either unable to utilise nitrate or would exhibit reduced growth on this nitrogen source. However, the fact that no G6PD mutants have been found among the nitrate non-utilising mutants suggests that such mutations are in fact lethal. How does <em>A. nidulans</em><em>gsd</em> A expression respond to NADPH consuming processes other than nitrate utilisation? Are these responses also regulated at the transcriptional level and if so which transacting factors are involved? What is the function of the individual <em>gsd</em> A-boxes in <em>A. niger</em> and <em>A. nidulans</em> ? Why do the regions containing the <em>gsd</em> A-boxes differ functionally, despite the obvious homology? Does titration of NIRA rely occur in <em>A. nidulans</em><em>gsd</em> A cotransformants? In any case these questions demonstrate the necessity for further research!
Redoxenzymen : daar zit wat in
Laane, N.C.M. - \ 1996
Wageningen : Landbouwuniversiteit Wageningen - 25
oxidoreductasen - enzymen - oxidatie - colleges (hoorcolleges) - compact discs - binding (scheikundig) - oxidoreductases - enzymes - oxidation - lectures - bonding
Assessment of cytochrome P450 1A in dab as biomarker of exposure to polychlorinated biphenyls and related compounds
Sleiderink, H.M. - \ 1996
Agricultural University. Promotor(en): J.H. Koeman; J.P. Boon. - S.l. : Sleiderink - 128
polychloorbifenylen - pleuronectidae - schol - pleuronectiformes - oxidoreductasen - cytochroom p-450 - tracer technieken - tracers - metabolisme - noordzee - limanda limanda - biologische indicatoren - ecotoxicologie - bioaccumulatie - polychlorinated biphenyls - plaice - oxidoreductases - cytochrome p-450 - tracer techniques - metabolism - north sea - biological indicators - ecotoxicology - bioaccumulation
<br/>The objective of this thesis was to investigate whether the CYP1A response in dab could be used as a biological marker of exposure to PCBs and related compounds in monitoring programmes in the southern North Sea. Only because of its wide occurrence in the North Sea, dab is already playing an important role in international contamination monitoring programmes, but no attempt has been made to investigate the suitability of the organism for monitoring purposes so far. During the field studies and laboratory experiments, emphasis has been laid on the study of PCBs, since they are by far the most important class of compounds capable of inducing CYP I A in the southern North Sea.<p>In chapter 2 the effects of maturity and sex on CYP1A expression in the field were investigated. Mature and juvenile dab of both sexes were collected from different areas of the southern North Sea with varying levels of PCB contamination. In all cases, muscle PCB concentrations were highest near the Dutch coast. The highest CYP1A levels, measured as EROD activity and CYP1A protein, were also found in this area for mature fish of both sexes in autumn. The same was found during the spawning season in winter for juvenile females and mature males. During this season gravid females showed significantly lowered contents of CYP1A protein and EROD activity compared to mature males and juveniles. Muscle PCB concentrations and both biochemical parameters were positively correlated for mature males, and not for other groups of dab, during both seasons. It was concluded that the sensitivity of CYP1A induction in dab as a biomarker for PCBs and related compounds was highest in mature males.<p>In chapter 3 it was shown that bottom water temperature differences of up to 10°C, occuring between stratified and vertically mixed areas during spring and summer, have a strong effect on CYP1A expression in dab. Highly elevated CYP1A levels were observed in mature male dab collected from off-shore stations with low bottom water temperatures due to stratification whereas considerably lower CYP1A levels were observed at stations with higher water temperatures in vertically mixed areas, including coastal stations. Statistical analyses of the data indicated that water temperature was inversely related to CYP1A levels, whereas PCB concentrations showed a positive correlation with CYP1A levels. The effect of water temperature, however, dominated over the effect of PCB contamination. A laboratory study confirmed that EROD activity was inversely proportional to water temperature. Furthermore, it was shown that differences in the nutritional status of dab, reflected by the condition factor, also obscured the effects of PCB contamination on CYP1A levels during these seasons.<p>Because it was demonstrated that spring and summer are less suitable seasons for monitoring purposes and next to this, dab in the southern North Sea show considerably more migration during the spawning period in winter, it was investigated whether the most suitable period for annual monitoring would be the late autumn. Chapter 4 describes a final field study during which differences in interfering natural factors, like water temperature, condition factor of the fish and percentage lipid in muscle tissue proved to be minimal. The results showed that the CYP1A induction response in male dab was strong enough to separate the Dutch coastal area from three more pristine offshore areas of the North Sea. Thus, the general conclusion of all field studies is that the late autumn indeed offers the best conditions to investigate the relation between environmental contamination with PCBs and related compounds, since disturbing influences of factors other than PHAH contamination are minimal.<p>The induction capacity of CYP1A by PCBs has been studied in laboratory experiments. Chapter 5 describes an experiment in which mature female dab were dosed with 1 mg of the technical PCB mixture Clophen A40 every 6 weeks, with a maximum of three doses per fish. Induction of CYP1A was observed following this Clophen A40 exposure. Biochemical effects were related to increases in concentrations of total PCBs and specific congeners and consequently to the corresponding toxicity equivalencies of these PCBs (CB-TEQs) in muscle. In all treated groups the EROD activity, CYP 1 A protein and total CYP 1 A levels were higher than those of the control groups. The maximum for these biochemical parameters was already reached after the first single dose, although the CB concentrations in muscle tissue increased further after administration of a second and third dose. It was concluded that the CYP1A system of dab is sensitive towards PCBs.<p>Since PCB congeners differ in potency to induce CYP1A the biochemical effects of several non- and mono- <em>ortho</em> substituted CB congeners were investigated. Exposure to the non- <em>ortho</em> chlorinated CB77 resulted in induction of CYP1A (chapter 6). On the contrary, exposure to three different mono- <em>ortho</em> chlorinated CB congeners and the non- <em>ortho</em> chlorinated CB126 did not result in increases of CYP1A levels (chapter 7). For CB126 this is in sharp contrast with earlier studies in fish where this congener was found to be a potent inducer of CYP1A. A large inter-individual variation in especially the reference group was held partly responsible for this. Exposure to CB 126 even caused a dose-dependent inhibition of EROD activity at higher concentrations.<p>In chapter 6 the influence of temperature on the temporal induction pattern of CYP1 A was investigated. Mature males were exposed to a single dose of a non- <em>ortho</em> chlorinated CB congener (CB77). The fish were acclimated at two different temperatures (10 and 16°C) and kept at these temperatures for a period of 40 days. At both temperatures, CYP1A protein and EROD activity were induced 40 days after dosing. Maximum responses of both EROD activity and CYP1A protein for the warm-acclimated fish were observed 5 days after treatment. For the cold-acclimated fish a slow, progressive elevation for both biochemical parameters was observed and maximum responses were measured 40 days after treatment. In the control groups EROD activity and CYP1A protein levels were higher in the cold-acclimated fish, which is in accordance with the study described in chapter 3. In the dosed groups, however, absolute biochemical levels were equally high at 40 days after treatment. It is therefore concluded that the magnitude of induction was higher in warm- acclimated fish.<p>Since CYP1A is a biomarker of exposure it does not provide knowledge of adverse (toxic) effects. The question can thus be raised whether enzyme induction reflects deleterious effects on dab. In the study where dab were exposed to Clophen A40 (chapter 5), no effects on reproductive parameters were found (Fonds <em>et al.</em> , 1995). It is very likely that the concentrations of contaminants needed for effects at higher levels of biological organisation will seldom be reached in the marine environment.<p>From the present research, it has become clear that dab is a suitable organism to monitor hepatic CYP1A indicating exposure to PHAHs, in the North Sea. Dab has shown to be sensitive towards PCBs. However, when applying the CYP1A measurement in dab, several interfering co- factors, like seawater temperature, maturity and sex, have to be taken into account. As a consequence, monitoring with mature males in the autumn seems to be preferable. With respect to monitoring programmes, the CYP1A measurement could very well serve as a quick and cheap screening method for contamination of the aquatic environment. The joint impact of a whole group of chemicals, the PHAHs, on CYP1A can be assessed. Although there is ample evidence for the existence of a dose-response relationship between PHAHs and CYP1A direct linear correlations will not always be found in the environment. For instance, concentrations of PAHs in the North Sea are too low to induce CYP1A in dab. Therefore measurement of CYP1A can never fully replace chemical analyses, but it can give complementary information about inducing PHAH compounds. Application of CYP1A seems to be useful to discrimate between estuarine and/or coastal areas and the open North Sea, which implies that increased concentrations of PHAHs reaching the marine environment via the outflow of contaminated rivers can be detected.
Superclusters : a search for novel structures and functions of biological iron-sulfur clusters
Arendsen, A.F. - \ 1996
Agricultural University. Promotor(en): C. Veeger; W.R. Hagen. - S.l. : Arendsen - ISBN 9789054855958 - 101
oxidoreductasen - ijzer - zwavel - oxidoreductases - iron - sulfur - cum laude
<br/>Iron sulfur (Fe-S) proteins are found in a variety of organisms. They usually function in electron transport, but they may also be involved in other functions like gene regulation and Lewis acid catalysis. The structure and spectroscopic properties of Fe-S clusters holding one, two, three, or four iron atoms is known to a great extent. These 'common' clusters share some basic properties. Firstly, they contain not more than four iron atoms. Secondly, despite the fact that they may contain more than one iron atom, they exist in two (physiological) redox states only. Thirdly, they are characterized by a low electron spin, i.e. they are usually S = 1/2. However, there is a strong indication that Fe-S clusters exist which do not obey these general rules. These clusters may hold more than four iron atoms, they are usually high-spin (S ≥3/2), and they may exist in more than two redox states. Because of these properties these clusters are referred to as superclusters. When I started this research project, six potential systems were proposed to contain larger (≥4Fe) or uncommon (WFe3S4) Fe-S clusters. These enzymes are involved in (multi-electron) redox catalysis:<p>1. Nitrogenase<br/>2. Fe-only hydrogenase<br/>3. Dissimilatory sulfite reductase<br/>4. carbonmonoxide dehydrogenase<br/>5. Prismane protein<br/><em>6. Pyrococcus furiosus</em> aldehyde oxidoreductase<p>The aim of my thesis is to study physical, chemical, and biological properties of multi- electron transferring enzymes, in a quest for possible new structures and functions of biological Fe-S clusters.<p>Chapter 2 describes the purification and characterization of a dissimilatory sulfite reductase from <em>Desulfosarcina variabilis.</em> The enzyme belongs to the class of desulforubidins, as was deduced from its UV/vis absorption spectrum. It is a a <sub><font size="-2">2β2γ2</font></sub> hexamer of ≈208 kDa, and it was found to contain ≈15 Fe and ≈19 S <sup><font size="-2">2-</font></SUP>. The oxidized enzyme exhibited S = 9/2 Fe-S EPR signals (g = 16 <em>).</em> Similar signals have previously been found in <em>Desulfovibrio vulgaris</em> (Hildenborough) desulfoviridin by Pierik and Hagen, who suggested the presence of a larger Fe-S cluster. With the finding of similar very high spin signals also in <em>D. variabilis</em> desulforubidin, it appears that the presence of a S = 9/2 Fe-S (super) cluster is common in all dissimilatory sulfite reductases. The sirohemes in <em>D. variabilis</em> desulforubidin were found to be fully metalated, and none of the Fe-S EPR signals gave indication for dipolar and/or exchange coupling with siroheme. These observations are interpreted as supportive evidence against the previously proposed model of a bridged cubane/siroheme as the active site for dissimilatory sulfite reductases.<p>The extreme hyperthermophile <em>Pyrococcus furiosus</em> contains a NiFe hydrogenase which not only reversibly oxidizes hydrogen but also reduces elemental sulfur (S <sup><font size="-2">0</font></SUP>) to H <sub><font size="-2">2</font></sub> S. The Archaeon was succesfully grown in a 200 l fermentor at 90°C on potato starch, and the hydrogenase could be purified aerobically without loss of activity. In contrast to previous reported data the enzyme was found to contain 17 Fe, 17 S <sup><font size="-2">2-</font></SUP>, and 0.74 Ni. Three EPR signals were found; a near-axial (g = 2.02, 1.95, 1.92) S = 1/2 signal ( <em>E</em><sub><font size="-2">m,7.5</font></sub> = - 303 mV) indicative of a [2Fe-2S] <sup><font size="-2">(2+;1+)</font></SUP>cluster, a broad spectrum of unknown origin (g = 2.25, 1.89; <em>E</em><sub><font size="-2">m,7.5</font></sub><em></em> = -3 10 mV), and a novel rhombic S = 1/2 EPR signal (g = 2.07, 1.93, 1.89) reminiscent of a [4Fe-4S] <sup><font size="-2">(2+;1+)</font></SUP>cluster. This rhombic signal appears with a reduction potential of <em>E</em><sub><font size="-2">m,7.5</font></sub><em></em> = -90 mV, and disappears at <em>E</em><sub><font size="-2">m,7.5</font></sub><em></em> = -328 mV. The latter observation suggested that this cluster is capable of taking up two electrons, and, therefore, that it is a supercluster. However, it is hypothesized that the disappearance of the signals at low potential is caused by magnetic interaction of the rhombic g = 2.07 signal with a third paramagnet, resulting in a broad interaction signal. Hence, there is no indication for the presence of a supercluster in <em>P.</em><em>furiosus</em> NiFe hydrogenase (chapter 3).<p>In the NiFe hydrogenases of several organisms as well as in the Fe-only hydrogenases of <em>Megasphaera eldenii</em> and <em>Desulfovibrio vulgaris</em> (Hildenborough) novel Fourier transform infrared (FTIR) detectable groups were found. The bands occur in the region of 2100-1800 cm <sup><font size="-2">-1</font></SUP>, which corresponds to stretching vibrations of polar triple bonds, metal hydrides, or asymetrically coupled vibrations of two adjacent double bonds. The position of these bands shifted upon oxidation and reduction of the enzymes. FTIR bands in this region were not detected in a large control group of Fe-S and/or nickel containing proteins including a metal-free hydrogenase. The FTIR groups in NiFe hydrogenases are assigned to the three unidentified small non-protein ligands that coordinate the Fe as observed in the X-ray structure of <em>Desulfovibrio gigas</em> NiFe hydrogenase. Thus far, the structural difference between NiFe- and Feonly hydrogenases had been thought to reside in the absence or presence, respectively, of a novel Fe-S cluster (H-cluster) which is proposed to be the site of hydrogen activation. The finding of similar FTIR groups in both Fe-only and NiFe-hydrogenases might suggest that the hydrogen-activating site of both classes of hydrogenases encompasses of a bimetallic center involving a low spin Fe ion with FTIR- detectable groups.<p>During the purification of several proteins from <em>Desulfovibrio vulgaris</em> strain Hildenborough a yellowish fraction eluted from the first anion exchange column at high NaCl concentration. It was observed that this fraction absorbed strongly at 260 nm. Attempts were made to purify the protein. The protein turned out to be a ferredoxin, as concluded from its size (a homodimer of subunits, each of 7.5 kDa), its pi (3.9) and its EPR spectrum in the reduced state, indicating the presence of a [4Fe-4S] <sup><font size="-2">(2+;1+)</font></SUP>cluster. The protein was associated with RNA having a typical size of 9-17 nucleotides. Hybridization experiments with extracted, radiolabeled RNA and digested <em>D. vulgaris</em> genomic DNA indicated that the ferredoxin binds either to total RNA or specifically to rRNA. The suggestion is made that <em>D. vulgaris</em> ferredoxin may not be a redox protein, but that it may have a regulatory function. This suggestion is supported by the unusually high standard reaction entropy of reduction of -230 J <strong><sup><font size="+1">.</font></SUP></strong> K <sup><font size="-1">-1</font><strong><font size="+1">.</font></strong></SUP>mol <sup><font size="-1">-1</font></SUP>. This would be the first prokaryotic Fe-S protein known to function in translation regulation.<p>In chapter 6 an EPR/redox study is presented on the tungsten-containing aldehyde oxidoreductase (AOR) from the hyperthermophile <em>Pyrococcus furiosus.</em> The enzyme had previously been suggested to hold a [WFe3S4] cluster. Highly active AOR could be obtained by rapid, anaerobic purification (i.e. within two days). Only active enzyme was used for this study. The fully reduced enzyme exhibited a mixture of S = 1/2 and S = 3/2 Fe-S EPR signals. Oxidized AOR afforded signals typical for W <sup><font size="-2">5+</font></SUP>(g = 1.982, 1.953, 1.885). Shortly after this research project started the X-ray structure of <em>P.</em><em>furiosus</em> AOR was elucidated by Chan <em>et al.,</em> who showed that the enzyme contains one [4Fe-4S] cluster and one tungsten cofactor per subunit. Our data are in agreement with the crystal structure, which excluded the possibility of a [WFe3S4] cluster. Such a cluster would have been a completely novel Fe-S cluster. The W <sup><font size="-2">5+</font></SUP>spectrum could be simulated using <sup><font size="-2">183</font></SUP>W hyperfine splitting constants A <sub><font size="-2">xyz</font></sub> of 7.7, 4.5, and 4.2 mT. A reduction potential <em>E</em><sub><font size="-2">m,7.5</font></sub> = +180 mV was determined for the couple W <sup><font size="-2">4+</font></SUP>/W <sup><font size="-2">5+</font></SUP>. Given the low reduction potential of the substrate, it is suggested that the biologically relevant redox chemistry may not not be located on the tungsten, but rather on the pterin cofactor.<p>The prismane proteins of <em>Desulfovibrio vulgaris</em> (Hildenborough) and <em>Desulfovibrio desulfuricans</em> ATCC 27774 are proposed to contain a [6Fe-6S] cluster. A similar cluster has been proposed to be present in the active site of Fe-only hydrogenases. Unfortunately, crystallographic evidence for the presence of [6Fe-6S] clusters is still lacking. Several attempts were made to crystallize the <em>D. vulgaris</em> (H) protein in our lab, but these were all unsuccessful. Eventually, high quality crystals were obtained in Daresbury, U.K. in collaboration with prof Lindley. Crystals grew within four days, and grew to a maximum size of 0.7 mm within ten days. The resolution of the diffraction pattern extends to 1.7 Å. The unit cell is orthorhombic, with spacegroup P2 <sub><font size="-1">1</font></sub> 2 <sub><font size="-1">1</font></sub> 2 <sub><font size="-1">1</font></sub> . The unit cell will readily hold four molecules of molecular mass of 60 kDa, with a solvent content of approximately 48%.<p>Generally, I looked for superclusters in the following multi-electron transferring enzymes: dissimilatory sulfite reductase, hydrogenase, and <em>P.</em><em>furiosus</em> aldehyde oxidoreductase. For <em>Desulfosarcina variabilis</em> dissimilatory sulfite reductase I found indication for the presence of a supercluster, whereas for hydrogenases the finding of novel FTIR resonances suggest a unique metal structure to be part of the active site. No indication was found for the presence of a supercluster in <em>Pyrococcus furiosus</em> aldehyde oxidoreductase, but the enzyme showed to be an interesting case for the study of biological tungsten. An apparent supercluster in <em>Pyrococcus furiosus</em> NiFe hydrogenase turned out to be most likely a [4Fe-4S] cluster. An RNA-binding ferredoxin from <em>Desulfovibrio vulgaris</em> (H) may be the first example of a prokaryotic, gene regulating Fe-S protein. Finally, elucidation of the crystal structure of the prismane protein from <em>Desulfovibrio vulgaris</em> (Hildenborough) will be a major step towards the development of the concept of larger Fe-S clusters.
Characterization of redox proteins using electrochemical methods
Verhagen, M. - \ 1995
Agricultural University. Promotor(en): C. Veeger; W.R. Hagen. - S.l. : Verhagen - ISBN 9789054853800 - 145
oxidoreductasen - cytochromen - elektrochemie - oxidoreductases - cytochromes - electrochemistry
<p>The use of electrochemical techniques in combination with proteins started approximately a decade ago and has since then developed into a powerfull technique for the study of small redox proteins. In addition to the determination of redox potentials, electrochemistry can be used to obtain information about the kinetics of electron transfer between proteins and about the dynamic behaviour of redox cofactors in proteins. This thesis describes the results of a study, initiated to get a better insight in the conditions necessary to obtain electron transfer between solid state electrodes and proteins.<p>Flavin Adenine Dinucleotide (FAD) is the subject of chapter 2. The electrochemical behavior of this cofactor, which is present in some flavoproteins, appeared to be dependent on its solution concentration. At concentrations of 1 μM the voltammetry showed all the characteristics of a species adsorbed to the surface. At a thousandfold higher concentration the voltammetry became completely diffusion controlled. From experiments at intermediate concentrations it was concluded that part of the FAD molecules adsorb to the electrode. Furthermore, it was shown that electron transfer between the molecules in solution and the electrode can only take place through the adsorbed molecules, which act as mediators. A comparison with results obtained with a 2 [4Fe-4S] ferredoxin from <em>Megasphaera elsdenii</em> suggested that, under certain conditions, a similar mechanism of selfmediation can be valid for proteins.<p>The results of a study of cytochrome <em>c</em> 553 from <em>D. vulgaris</em> (H) <em></em> are presented in chapter 3. The cytochrome was characterized by cyclic voltarnmetry and the same technique was used to determine the rate of electron transfer between the cytochrome and the Fe-hydrogenase from the same organism. The results indicated that the cytochrome was a physiologically competent redox partner dependent on the in vivo function of the hydrogenase. Since the function of the hydrogenase is still an issue of debate it is not known whether this new electron transfer pathway has physiological relevance.<p>The reinvestigation of the protein desulfoferrodoxin from <em>D. vulgaris</em> (H) is described in chapter 4. This protein was reported to contain two iron atoms one of which was coordinated by four cysteine residues in a distorted tetrahedron. By comparison with model compounds using EPR spectroscopy and by using cyclic voltarnmetry at different pH values it was shown that this is very unlikely. Instead it is proposed that the iron atom is coordinated in a pentagonal bipyramid (surrounded by 5 ligands in a plane and 2 ligands perpendicular to and on both sides of this plane). Furthermore the controversy about the protein having a mixed N-terminus was elucidated and it was established that the protein was a homodimer instead of the reported monomer.<p>The conditions necessary for the use of direct electrochemistry to study small redox proteins become more and more established. The application of this technique to enzymes is, however, not straightforward, The reason for this is not clear, but one possibility is that a large enzyme adsorbs more easily to the electrode than a small protein. Another possible explanation is that the redox cofactors in enzymes are shielded more by the protein matrix. In order to circumvent this latter problem we tried to establish conditions for the electron transfer between cytochrome P- 450 from <em>P. putida</em> and glassy carbon electrodes. This bacterial cytochrome P-450 has a ferredoxin as a physiological electron donor and has therefore a docking place where the electrons can enter the enzyme. When using the right conditions it should be possible to let the electrode be the "substrate" for the enzyme. Unfortunately the enzyme adsorbed to the electrode and the obtained value for the potential was much more positive than the literature value. An EPR redox titration of the cytochrome P-450 indicates that the literature value needs a correction. However, there still remains a difference between the value obtained from the titration in homogeneous solution and the value determined electrochemically.<p>Recent reports about electrochemical characterization of superoxide dismutase from bovine erythrocytes incited the study described in chapter 6. The conditions used in the reported electrochemical experiments were rather extreme <u>i.e.</u> low pH. EPR monitored redox titrations of the enzyme at different pH values indicated that the oxidation and reduction at low pH values is not reversible. Furthermore, it was found that the reported potentials at pH 7.0 needed to be corrected. A redox titration was also performed with the iron enzyme from <em>E. coli</em> as a comparison with the copper zinc containing enzyme. After reduction, however, it was not possible to reoxidize the enzyme again indicating that the redox reaction is not reversible. This can explain the huge differences in potentials reported so far in the literature.<p>The use of a redox active promotor can give some insight in its mechanism of action. The lanthanide europium proved to be a potent promotor of rubredoxin. The latter is a small purple redox protein containg a single iron coordinated to 4 cysteine residues. At high pH values the reduction and oxidation of rubredoxin is readily obtained despite the fact that the europium ion does not show any reduction or oxidation anymore. This is not consistent with the models used to explain the promotor function of cations. These models all assume that the cation provides charge compensation and sandwiches both between the protein and the electrode as well as between different protein molecules. The results from this study are presented in chapter 7.<p>A great advantage of electrochemistry using glassy carbon electrodes is that it is possible to vary the potential between approximately -1 V and +0.8 V. This makes it possible to study redox couples with potentials more negative than the commonly used chemical reductants like dithionite or titanium citrate. This led to the discovery of the superreduction of the Rieske cluster in the soluble fragment of the <em>bc1</em> complex of beef heart as described in chapter 8. This protein contains an [2Fe- 2S] cluster with a redox potential of + 312 mV versus SHE. At low potential (-840 mV versus SHE) it is possible to reduce this cluster with a second electron. The physiological relevance of this superreduced state is not clear but its characterization can give insight in the mechanism of multiple electron transfer by iron sulfur clusters.<p>The final two chapters are used to describe the biochemical and spectroscopic characterization of two proteins from <em>D. vulgaris</em> . Adenylyl sulphate reductase <em>(AdoPSO <sub>4</sub></em> reductase) is an enzyme which is involved in the sulfate respiration of the bacterium. It reduces the activated sulfate <em>(AdoPSO <sub>4</sub></em> ) to AMP and sulfite. Literature reports indicated that the protein contained one FAD and two [4Fe-4S] clusters. The presence of two clusters was based on the observation of a complicated EPR spectrum which indicates interaction between two paramagnetic centers. In our studies however this "interaction" spectrum only appeared when the enzyme solution was at low ionic strength. Upon raising the ionic strength with an inert salt like NaCl the complicated EPR spectrum changed into a spectrum of a single S-1/2 species. This indicated that the interaction between the paramagnetic centers was intermolecular rather than intramolecular. This observation led us to propose that <em>AdoPSO <sub>4</sub></em> reductase contains one FAD and one Fe-S cluster. Since the average metal analysis showed the presence of 6 iron atoms and 5 acid labile sulfur atoms it was proposed that the Fe-S cluster may have an iron content greater than 4.<p>Chapter 10 describes the results of a study of high molecular weight cytochrome <em>c</em> . This protein resides in the periplasmic space of <em>D. vulgaris</em> and contains sixteen hemes. Its function is up till now unknown. In previous reports midpoint potentials were reported for the different hemes based on single scan differential pulse voltammetry. These values might be erroneous due to the absence of reversibility. Indeed an equilibrium redox titration monitored by EPR indicated that the reported values were incorrect. Furthermore, it was not possible to reproduce the reported voltammograms. This confirmed our observation that the electrochemistry of large proteins or enzymes is often difficult to interpret and difficult to reproduce. It is also a good example of how important it is to check whether or not reversibility applies during electrochemical experiments.
On the role of phospholipids in the cytochrome P450 enzyme system
Balvers, W.G. - \ 1994
Agricultural University. Promotor(en): C. Veeger; Ivonne Rietjens. - S.l. : Balvers - 219
enzymen - oxidoreductasen - cytochroom p-450 - membranen - toxische stoffen - xenobiotica - enzymes - oxidoreductases - cytochrome p-450 - membranes - toxic substances - xenobiotics
<p>The cytochrome P450 enzyme system is involved in the metabolism and elimination of an almost unlimited number of endogenous and exogenous substrates. Biotransformation by cytochromes P450 plays a role in the conversion xenobiotics into more hydrophilic products. Generally, this process of biotransformation in which cytochrome P450 reactions take part, leads to elimination of the xenobiotic through urine and / or faeces although in some cases this process can also lead to the formation of more toxic metabolites. Because of its significant role in the conversion of numerous endogenous and exogenous compounds, a complete understanding of the cytochrome P450 enzyme system is of importance in toxicology, pharmacology, anesthesiology, pathology and other related biomedical fields.<p>Twenty-five years ago the phospholipids of the membrane of the endoplasmatic reticulum, to which the enzyme system is bound, appeared to play an important role in the <em>in vitro</em> cytochrome P450 enzyme system. Although the role of the membrane(phospholipids) in the cytochrome P450 system has been intensively studied since then, it has not resulted in a unanimous conclusion. The goal of this thesis was therefore, to gain further insight into the role of the membrane and membrane phospholipids in the cytochrome P450 system. Attention is thereby not only paid to the effects of the membrane and the phospholipids on cytochrome P450 enzymes but also to the effects on another important protein component <em>of</em> the enzyme system, namely NADPH-cytochrome reductase.<p>In the first two chapters the cytochrome P450 enzyme system and the membrane(phospholipids) of <em></em> the endoplasmatic reticulum are described respectively. In <strong>chapter 1A</strong> the structural and catalytical properties of the cytochrome P450 enzyme system are briefly discussed. Special attention is paid to the occurence, multiplicity, induction, the structure of the individual protein components of the enzyme system and the catalytic cycle of cytochromes P450. The individual steps in this catalytic cycle are discussed in detail.<p><strong>Chapter 1B</strong> deals with the structural aspects <em>of</em> the membrane phospholipids and the membrane of the endoplasmatic reticulum. In addition a brief description of the different types of reconstituted systems used in this thesis is given. Finally, the effects of phospholipids on the cytochrome P450 enzyme system, reported up to now in the literature, and various current hypotheses for the stimulating effect of phospholipids are discussed briefly.<p>In <strong>chapter 2</strong> results are described that characterise the sensitivity of microsomal and isolated cytochrome P450 IA1 and IIB1 for an organic hydroperoxide; cumene hydroperoxide (CuOOH). These data provide information on the difference in the way of membrane incorporation of these two cytochrome P450 enzymes. Up to now, very little attention has been paid to possible differences in sensitivity of cytochromes P450 to conditions of oxidative stress. A difference in sensitivity for (hydro)peroxides between different forms of cytochrome P450, as demonstrated with CuOOH in the present study, can be of importance from a toxicological point of view. Especially in cases in which conversion by one cytochrome P450 enzyme results in detoxification of the substrate whereas another cytochrome P450 enzyme causes bioactivation of the substrate, a difference in sensitivity for conditions of oxidative stress can then result in a shift in the metabolite pattern.<p>Cytochrome P45 0 IIB1, embedded in the microsomal membrane is more sensitive towards CuOOH treatment than microsomal cytochrome P450 IA1. Purification of these enzymes and reconstitution in a system in which the proteins remain soluble results in a disappearance of the difference in CuOOH sensitivity between the two cytochrome P450 enzymes. Upon incorporation of cytochrome P450 IA1 and IIB1 into an artificial membrane, cytochrome P450 IIB1 again appears to be more sensitive towards CuOOH than cytochrome P450 IA1. Furthermore, the EC-50 values (effective cumene hydroperoxide concentration which causes 50% inhibition of cytochrome P450 dependent activities) in the microsomal and membrane incorporated reconstituted systems are comparable. Based on these results it is concluded that (1) the difference in sensitivity between cytochrome P450 IA1 and IIB1 towards treatment with CuOOH originates from a difference in the way these cytochrome P450 enzymes are incorporated into the membrane, that (2) the purification procedure does not affect the parameters determining the way of incorporation of the protein into the membrane and that (3) the way of membrane incorporation of cytochrome P450 enzymes in microsomal and reconstituted systems is comparable.<p><img src="/wda/abstracts/i1817_1.gif" height="904" width="600"/><br/>In <strong>chapter 3</strong> the effects of changes in the fatty acyl moiety of phosphatidylcholine (PC) from dilauroyl (di 12:0) to distearoyl (dil8:0) on the kinetics of reconstituted cytochrome P450 IA1 and IIB1 were investigated. So far, studies on the effect of phospholipids on the kinetics of cytochrome P450 dependent reactions have focussed on one P450 enzyme or one phospholipid. Furthermore, mainly the effect on the activity at non-saturating substrate concentrations was investigated without paying much attention to the effect on the kinetic parameters K <sub>m</sub> and V <sub>max</sub> , of a cytochrome P450 catalysed reaction. An effect of phospholipids on for example the K <sub>m</sub> could be of considerable importance especially because in living organism the substrate concentrations will generally be low. Furthermore, a different effect of phospholipids on the substrate's apparent Km of different cytochrome P450 enzymes - converting the substrate to different metabolites - might not only affect substrate conversion rates but also the metabolite pattern.<p>The results presented in chapter 3 demonstrate that the V <sub>max</sub> of the cytochrome P450 dependent O-dealkylation of alkoxyresorufins and ethoxycoumarin for both cytochrome P450 IA1 and IIB1 is two times higher in the PC di 12:0 system compared to the PC di 18:0 system. The effect of a change in the fatty acyl moieties on the Km of the xenobiotic substrate however, appeared to be different for the two cytochrome P450 enzymes. For cytochrome P450 IA1 the K <sub>m</sub> appeared to be lower in the PC di 12:0 system compared to the PC dil8:0 system whereas for cytochrome P450 IIB1 the K <sub>m</sub> was lowest in the PC di l8:0 system. Additional results demonstrated that the kinetic parameters were dependent on the PC : P450 ratio and that changing this ratio affected the kinetic parameters of cytochrome P450 IA1 and IIB1 in a different way.<p>The reason for the differential effect on the substrate apparent K <sub>m</sub> was further investigated in a series of experiments in which the effect of PC di 12:0 and PC di l8:0 on individual steps of the catalytic cycle of cytochrome P450, like substrate binding, oxygen binding and rate of electron transfer, was studied. From these experiments it was concluded that the higher V <sub>max</sub> in the PC di 12:0 system, observed for both cytochrome P450 enzymes, was at least in part due to the higher affinity of cytochrome P450 for NADPH-cytochrome reductase in the PC di 12:0 system. Furthermore, these experiments demonstrated that the different effect of a change in the fatty acyl moieties of PC on the K <sub>m</sub> of cytochrome P450 IA1 and IIB1 did not result from a different effect on substrate binding, oxygen binding and rate of electron transfer. This means that the differential effect on the K <sub>m</sub> must result from an effect on one or more of the other steps in the catalytic cycle such as reductive oxygen splitting, substrate conversion and / or product release.<p>The results show that the effect of a change in the type of PC and or the PC : P450 ratio on the kinetic parameters, K <sub>m</sub> and V <sub>max</sub> , is dependent on the cytochrome P450 enzyme used in the reconstitution. Furthermore, in contrast to what is generally assumed and based on results under non- saturating substrate conditions [1 -4], the addition of PC appears to result for some cytochrome P450 enzymes in a decrease of the V max in the reconstituted system. The results in chapter 3 also demonstrate that this is not reflected in lower but - in contrast - in higher conversion rates at non- saturating substrate concentrations (Figure 9.1), because the K <sub>m</sub> is decreased simultaneously.<p>In <strong>chapter 4</strong> the existence of a preference - with respect to binding - of cytochrome P450 IIB1 for phospholipids with certain headgroups or fatty acyl moieties was investigated. The existence of "boundary" phospholipids (phospholipids which bind to cytochrome P450 with specificity and high affinity) for microsomal cytochrome P450 has been a topic for several studies. Nevertheless, little is known about this subject and unanimous conclusions have not been reached. It has been suggested that the composition of the membrane in the direct vicinity of cytochromes P450 is different from the rest of the membrane [5] and that specific interactions exist between cytochrome P450 and phosphatidylethanolamine (PE) [61 and phosphatidic acid (PA) [7].<p>The results in chapter 4 show that the apparent binding constant (K <sub>d</sub> ) of a cytochrome P-450 IIB1 - phospholipid complex is dependent on the degree of unsaturation of the phospholipid side chains; demonstrating a decrease in the Kd with increasing degree of unsaturation, but independent of the length of the acyl chains. In addition, the apparent Kd appeared to be dependent on the headgroup of the phospholipid molecule, showing a significantly higher K <sub>d</sub> for PE di 16:0 compared to PC di 16:0, PS di 16:0 and PI 16:0/18:1.<p>Translation of these results to the in vivo situation has to be done with caution because the results were obtained in a reconstituted system with isolated, solubilised cytochrome P450. In the membrane of the endoplasmatic reticulum other factors such as for example the presence of other proteins can play an additional important role in the interaction of cytochrome P450 with phospholipids. Furthermore, the effect of the length and the degree of unsaturation of the fatty acyl chains on the Kd was determined for PC. It remains to be established whether for phospholipids with different headgroups similar influences of the length an degree of unsaturation of the acyl chains are observed. Investigations in this direction are however, seriously hampered by the fact that series of pure molecular species of PE, PS and PI are not commercially available.<p>In <strong>chapter 5</strong> the existence of specific phospholipid : protein interactions for NADPH -cytochrome reductase was investigated. Compared to cytochrome P450 very little attention has been paid to possible interactions between phospholipids and NADPH-cytochrome reductase and possible consequences of such interactions for the cytochrome P450 system. NADPH-cytochrome reductase is a very important component of the cytochrome P450 enzyme system and the stimulating effect of phospholipids on the rate of cytochrome P450 dependent reactions may in part originate from an effect on NADPH-cytochrome reductase resulting in a more efficient electron transfer to the cytochromes P450.<p>Based on the results from 31P-NMR experiments and chemical analysis, it was concluded that NADPH-cytochrome reductase exhibits a preference for the negatively charged phospholipids phosphatidylserine (PS) and phosphatidylinositol (PI). In addition, experiments investigating the possible consequences of a special interaction of NADPH-cytochrome reductase with PS and PI demonstrated that (1) PS and PI had a significantly different effect on the DPH-PC dependent quenching of tryptophan fluorescence of NADPH-cytochrome redcutase compared to PE and PC and that (2) the V <sub>max</sub> of cytochrome P450 IIB1 dependent O-dealkylation of pentoxyresorufin in the presence of 1:1 mixtures of PS:PC and PI:PC were respectively higher and lower compared to the Vmax in the presence of a 1:1 mixture of PE:PC or PC alone. These phenomena might best be explained by a PS and PI induced specific change in the conformation of NADPH-cytochrome reductase. Regarding the fact that the specific interaction in both cases involves a negatively charged phospholipid suggest a possible role of the phospholipid charge. However, the fact that the effects of PS and PI on the V <sub>max</sub> of the cytochrome P450 catalysed reaction are different demonstrates that phospholipid charge cannot be the only factor.<p>In <strong>chapter 6</strong> the redox cycling of 7-alkoxyresorufins and the product of their metabolism by cytochrome P450, resorufin, by NADPH-cytochrome reductase is investigated. Redox cycling is a process in which a substrate is I-electron reduced, in this case by NADPH-cytochrome reductase. The electron is transfered to molecular oxygen and the substrate is returned to its initial state and can enter a new cycle. During this process reactive oxygen species are formed which can initiate lipidperoxidation and / or inactivate cytochrome P450. Especially in systems in which the NADPH-cytochrome reductase concentration is relatively high this process is a disturbing side-reaction, because it uses up reduction equivalents resulting only in the formation of reactive oxygen species which may cause protein inactivation and lipidperoxidation. In the reconstituted systems used in this thesis redox cycling can play an important role because the NADPH-cytochrome reductase : cytochrome P450 ratio is 15 to 30 times higher than in the in vivo situation. Furthermore, cytochrome P450 has been demonstrated to be very sensitive to lipidhydroperoxides - formed during lipidperoxidation - and reactive oxygen species.<p>The results of the present chapter demonstrate that at physiological pH alkoxyresorufins are much better substrates for redox cycling than resorufin. The inability of resorufin to stimulate redox cycling originates from the fact that at physiological pH resorufin exists mainly in its deprotonated form and this form is a much worse substrate for redox cycling than its protonated form. AM1 molecular orbital computer calculations demonstrated that the energy (E) of the lowest unoccupied molecular orbital (LUMO), i.e. the orbital into which the electron will be placed during redox cycling, of the deprotonated form is higher compared to the E <sub>LUMO</sub> of the protonated form. Furhermore, one-electron reduction of the protonated form appeared to be energetically favorable by 363.5 kJ/mol over one-electron reduction of the deprotonated form. In addition, the computer calculations demonstrated that the one electron reduced resorufin is most likely to become protonated at the O-atom of the intramolecular semiquinone imine moiety before reduction by a second electron. Finally, it was demonstrated that incorporation of NADPH-cytochrome reductase into an artificial membrane results in an increased redox cycling activity of resorufin compared to solubilized NADPH-cytochrome reductase. This was explained by an increase in the protonated form in the membrane either by (1) favored partitioning of the protonated form into the membrane or by (2) an effect of the membrane on the protonation equilibrium of resorufin in favor of the protonated form. This result points at the role of the membrane in concentrating apolar substrates of the cytochroom P450 : NADPH-cytochrome reductase svstem [8-10].<p>In chapter 7 the use of AM1 MO calculations in predicting the ability of compounds to stimulate redox cycling, as demonstrated in chapter 6, was further investigated. Therefore, in addition to resorufins, the redox cycling ability of 1,4-benzoquinones was investigated. Quinones are toxic compounds that are often used in chemistry. They are also used in pharmacology, for example as anticancer drugs because of their toxic character. The mechanism through which quinones exert their toxic effects is believed to involve the covalent binding of quinones to cellular nucleophilic macromolecules and /or the quinone catalysed process of redox cycling. 1,4- Benzoquinone has been demonstrated to redox cycle very poorly. In the literature, the poor redox cycling of 1,4-benzoquinone has been ascribed to a very high 1-electron reduction potential. The results from chapter 7 demonstrate however, that, at physiological pH, 1,4-benzoquinone is quickly 2-electron reduced by NADPH-cytochrome reductase to form 1,4- hydroquinone. Instead of transfering its electron on to molecular oxygen, the 1 -electron reduced semiquinone is protonated and subsequently reduced by a second electron. However, at pH>9 the 1,4-benzoquinone appears to be capable of stimulating redox cycling. Furthermore, the pH- and concentration-dependencies of redox cycling in a system with NADPH- cytochrome reductase between 1,4-benzoquinone and 1,4-hydroquinone are demonstrated to be similar. Based on these observations it was concluded that 1,4-benzoquinone is capable of redox cycling from its deprotonated, 2-electron reduced form at relatively high pH levels which ensures an adequate concentration of the deprotonated form The results from chapters 6 and 7 demonstrate the importance of the protonation / deprotonation equilibrium of the I- and 2-electron reduced forms in the redox cycling process.<p>In the paragraphs above the results of the study on the role of phospholipids in the cytochrome P450 : NADPH-cytochrome reductase system as it was executed for this PhD thesis are presented. Ever since 1968 [11] the role of phospholipids in this system has been a topic for numerous studies in which many questions concerning this role have been answered but many new questions have also been raised. Unfortunately, this thesis does not provide the answers to all these new questions because the cytochrome P450 system is too complex and the number of different phospholipids and the effects they induce is too large. Answering these questions will require many, many years of additional research. The goal of this thesis was to investigate certain aspects of phospholipids and the cytochrome P450 enzyme system to which up to now little attention has been paid in order to gain further insight into the role of phospholipids in the cytochrome P450 enzyme system.<p>Altogether, the results of the experiments in this thesis present some new insightsin the role of phospholipids on the cytochrome P450 enzyme system. Furthermore, additional evidence for already existing hypotheses of the stimulating effect of phospholipids are also presented. The conclusions of the present thesis can be summarized as follows.<br/>(1) Cytochromes P450 can differ in the way they are incorporated in the membrane which might result in differences in their sensitivity towards cumene hydroperoxide.<br/>(2) The way of incorporation of cytochrome P450 enzymes in microsomal and reconstituted systems is comparable.<br/>(3) Phospholipids influence the kinetic parameters, Km and Vmax, of cytochrome P450 catalysed reactions in reconstituted systems.<br/>(4) The result of the effect phospholipids on the kinetic parameters of cytochrome P450 dependent reactions is not the same for all P450 enzymes.<br/>(5) The affinity of cytochrome P450 for NADPH-cytochrome reductase in a reconstituted system is dependent on the fatty acyl moiety of the phospholipid added to the system.<br/>(6) Phospholipids can decrease the apparent Kd of cytochromes P450 for their xenobiotic substrates.<br/>(7) The apparent K <sub>d</sub> of cytochromes P450 for phospholipids is dependent on the headgroup of the phospholipid and the degree of unsaturation of the fatty acyl chains but independent of the length of the acyl chains of the PC molecule.<br/>(8) There is a specific interaction between NADPH-cytochrome reductase and the negatively charged phospholipids PS and PI.<br/>(9) The membrane functions as a place where apolar substrates can accumulate thereby decreasing the apparent K <sub>m</sub> .<br/>(10) The membrane causes a shift in the overall protonation equilibrium of the substrate towards the protonated form.<p>In addition to these conclusions a number of other interesting phenomena have been observed that have no bearing on the role of phopsholipids in the cytochrome P450 enzyme system but are also worth mentioning again.<br/>(1) The parameters determining the way of incorporation of cytochromes P450 in the membrane are not affected by the isolation procedure.<br/>(2) AM1 molecular orbital calculations is a useful additional tool in investigating the redox cycling capacity of chemicals.<br/>(3) The protonationequilibria of I- and 2-electron reduced compounds play an important role in their redox cycling ability.<p>Finally, regarding the results of the experiments presented in this thesis one final conclusion must be added. The effect of phospholipids on the cytochrome P450 enzyme system is dependent on many factors such as the cytochrome P450 form, the fatty acyl moiety and headgroup of the phospholipid, the P450 : reductase ratio and the phospholipid : P450 ratio. Therefore, for a complete understanding of the mechanism(s) of action of phospholipids in the cytochrome P450 enzyme system, a detailed investigation of the effects of all phospholipids (and mixtures of phospholipids) on all P-450 forms at several P-450 : reductase and phospholipid : P-450 ratio's is necessary. This requires a vast amount of work although some of this work has already been done in these last twenty- five years. Comparison of these results is , however, difficult because of the different conditions used in these studies. It is therefore, advisable to come, in analogy to the nomenclature of cytochrome P-450, to standardized conditions for research in order for the results of differentlaboratories to be compared.
Studies on the iron - sulfur clusters of hydrogenase, sulfite reductase, nitrogenase and the prismane protein
Pierik, A.J. - \ 1993
Agricultural University. Promotor(en): C. Veeger; W.R. Hagen. - S.l. : Pierik - 167
oxidoreductasen - nitrogenase - ijzer - zwavel - thiobacillus - sulfaat reducerende bacteriën - foto-elektronenspectroscopie - oxidoreductases - iron - sulfur - sulfate reducing bacteria - photoelectron spectroscopy - cum laude
<p>Iron-sulfur clusters are present in a large number of proteins. Sofar structures of four types of protein-bound iron-sulfur clusters have been determined by X-ray diffraction: rubredoxin-like, [2Fe-2S], [3Fe-4S] and [4Fe-4S] centers. The presence of any of these clusters in a protein can be predicted by comparison of spectroscopic properties. However a number of multiple-electron transferring enzymes, like the Fe-only hydrogenase, sulfite reductase and nitrogenase MoFe protein have enigmatic iron-sulfur clusters with spectroscopic properties unlike those of the known structures. These enzymes share a high iron and acidlabile sulfur content and the presence of superspin systems with S≥5/2. In this thesis biochemical and spectroscopic studies are presented on the above-mentioned iron-sulfur proteins and two unusual newly discovered iron-sulfur proteins, the 'prismane' protein and nigerythrin.<p>Chapter 2 summarizes new findings on the Fe-only hydrogenase and the redox properties of its cubanes and hydrogen activating iron-sulfur H-cluster. The hydrogenase, aerobically isolated from the sulfate-reducing bacterium <em>Desulfovibrio vulgaris</em> (Hildenborough) was shown to be composed of a mixture of high and low activity charge conformers, which can be isolated and discriminated by chromatographic and electrophoretic techniques. The redox properties of the rhombic S=1/2 EPR signals associated with the H-cluster of hydrogenase preactivated with dithionite or hydrogen were considerably more simple than those reported by Patil and coworkers for the (oxygen-insenstive) resting enzyme. Instead of sequential bell-shaped curves for the rhombic g=2.07 (g=1.96 and g=1.89) and g=2.11 (g=2.05 and g=2.00) EPR signals in reductive dye-mediated titrations, a simple behaviour with two redox states was observed both in reductive and oxidative titrations. The interconversion between the diamagnetic reduced and the oxidized redox state of the H-cluster exhibiting the g=2.11 S=1/2 EPR signal occurred at -307 mV. The bell-shaped nature and the occurrence of the g=2.07 rhombic EPR signal thus was due to the activation process of the H-cluster. By equilibration with H <sub>2</sub> /H <sup>+</SUP>of activated hydrogenase a midpoint potential of -330 mV was determined for the cubanes. A similar midpoint potential was observed in a dyemediated titration of a recombinant hydrogenase lacking the H-cluster. In these experiments no evidence for redox interaction between the two cubanes was seen.<p>In the course of extensive purification procedures of the Fe-hydrogenase it was recognized that an iron-sulfur protein with novel EPR spectroscopic properties occasionally contaminated hydrogenase preparations (Chapter 3). The as isolated form had a substoichiometric S=1/2 EPR signal with g=1.97, g=1.95 and g=1.90. Chemical analysis showed that, although such g-values are typical for Mo <sup>5+</SUP>= (or W <sup>5+</SUP>) no metals other than iron were present. The 'molybdenum'-like EPR spectrum disappeared both on reduction and oxidation. In the dithionite reduced form an almost stoichiometric S=1/2 EPR signal was observed. The g-values (g=2.00, 1.82 and 1.32) were reminiscent of those of prismane model compounds in the [6Fe-6S] <sup>3+</SUP>redox state. Therefore the protein was proposed to be a prismane-containing protein, in agreement with the chemical analysis indicating ≈6 Fe/protein. The occurrence of S=1/2 EPR signals in the as isolated state and dithionite reduced state could be explained by the assumption that the 'prismane protein' had four redox states: the fully reduced [6Fe-6S] <sup>3+</SUP>state with the fingerprint prismane signal, an [6Fe-6S] <sup>4+</SUP>redox state with unknown spin state (S=0 or integer), the [6Fe-6S] <sup>5+</SUP>state with the molybdenum-like S=1/2 EPR signal and the fully oxidized [6Fe-6S] <sup>6+</SUP>redox state (S=0 or integer).<p>The purification, chemical analysis and biochemical characterization of this 'prismane protein' are described in Chapter 4. The 'prismane' protein is a monomeric, cytoplasmic protein with a molecular mass of 52 kDa as estimated by sedimentation-equilibrium centrifugation. The protein contained 6.3±0.4 Fe and 6.2±0.7 S <sup>2-</SUP>per polypeptide. With polyclonal antibodies similar 'prismane' proteins were detected in <em>Desulfovibrio vulgaris</em> (Monticello) and <em>Desulfovibrio desulfuricans</em> (ATCC 27774). Using the N-terminal sequence and antibodies against this prismane protein Stokkermans and coworkers have sequenced the gene coding for this prismane protein as well as the homologous protein of <em>Desulfovibrio desulfuricans (ATCC</em> 27774).<p>Further spectroscopic evidence for a new iron-sulfur cluster and strong support for the presence of a prismane core is presented in Chapter 5. The discovery that the [6Fe-6S] <sup>5+</SUP>redox state exhibited a spin mixture of approximately stoichiometric S=9/2 and substoichiometric molybdenum-like S=1/2 EPR signals confirmed the earlier hypothesis of four redox states of the prismane protein. Dye-mediated redox titrations and the observation of a g=16 signal with increased intensity in parallel-mode EPR for the [6Fe-6S] <sup>4+</SUP>redox state completed the following scheme:<p><img src="/wda/abstracts/i1679_1.gif" height="96" width="600"/><p>Multiple frequency EPR spectroscopy of the S=1/2 EPR signals showed that additional broadening indicative of nitrogen ligation was present. The line broadening caused by enrichment of the prismane protein with <sup>57</SUP>Fe was in agreement with ≈6 Fe per cluster. Quantitative high-resolution Mössbauer spectroscopy of the <sup>57</SUP>Fe enriched prismane protein revealed that both in the [6Fe-6S] <sup>3+</SUP>and the [6Fe-6S] <sup>5+</SUP>form the iron ions were inequivalent. <em>A</em> 4:2 ratio of quadrupole doublets was observed. The quadrupole splitting and isomer shift of the four irons ions were relatively invariant to the redox change of the cluster, while the two apparently more ionic irons had a more pronounced change from Fe <sup>2+</SUP>to Fe <sup>3+</SUP>character. Mössbauer spectroscopy at low temperatures and with applied magnetic fields indicated that the four and two iron ions were present in the same magnetically coupled structure. This led to a model in which the prismane structure is composed of a central set of four iron ions with a more ionic iron ion on each side. The more ionic iron ions could correlate with the nitrogen ligation as inferred from EPR studies.<p>The unique EPR spectroscopic properties of the 'prismane protein' prompted investigation of dissimilatory sulfite reductase (desulfoviridin), a readily available iron-sulfur enzyme obtained during the isolation of Fe-hydrogenase from <em>Desulfovibrio vulgaris</em> (Hildenborough). The scrutiny for pure and electrophoretic homogeneous preparations of the desulfoviridin for EPR spectroscopic studies unexpectedly led to the discovery of a third, hitherto unrecognized 11 kDa subunit in this enzyme (Chapter 6). The γsubunit appeared to be tightly bound in the desulfoviridin complex for which a subunit composition of α <sub>2β2γ2</sub> was determined. N-terminal sequences and polyclonal antibodies against the α, βand γsubunits were obtained. The polyclonal antibodies allowed demonstration of the presence of homologous α, βand γsubunits in desulfoviridin-type dissimilatory sulfite reductases of three other <em>Desulfovibrio</em> species.<p>Chapter 7 delineates the redox and spectroscopic results on the siroheme and S=9/2 EPR signals of desulfoviridin. By summation of the S=1/2 and S=5/2 EPR signals of the siroheme group it was shown that only 20% of the siroheme groups were metallated. The midpoint potential for the Fe <sup>2+</SUP>/Fe <sup>3+</SUP>transition of the main species of the siroheme was -295 mV. No significant amounts of EPR signals of normal iron-sulfur clusters were observed. Instead, several novel EPR signals with g= 17, g= 15. 1, g= 11.7 and g=9.0 were found in the as isolated oxidized form of the protein. These EPR signals were from a paramagnet with S=9/2. <em>A</em> stoichiometry of approximately 0.6 spin per αβγwas estimated. In a reductive redox titration the S=9/2 EPR signals disappeared with E <sub>m</sub> =-205 mV. It was proposed that in the desulfoviridin -type dissimilatory sulfite reductase larger iron-sulfur clusters are present which give rise to the S=9/2 EPR signals. The demetallation of the siroheme and the S=9/2 EPR signals from an iron-sulfur cluster were in contradiction with the model of Siegel and coworkers for the sulfite reductase of <em>Escherichia coli,</em> in which coupling between a regular [4Fe-4S] <sup>2+</SUP>cubane and the Fe <sup>2+/3+</SUP>ion of the siroheme is proposed to explain spectroscopic properties.<p>In Chapter 8 the redox and EPR spectroscopic properties of the nitrogenase MoFe protein from <em>Azotobacter vinelandii</em> are described. By controlled oxidation with dye-mediated redox titrations the long lasting controversy on the spin and redox states of the oxidized P-cluster iron-sulfur centers was solved. It turned out that oxidation of the P clusters could lead to two consecutive redox states, P <sup>OX1</SUP>and P <sup>OX2</SUP>. On oxidation of the dithionite reduced P-clusters by two electrons (E <sub>m≈</sub> -307 mV) first the P <sup>OX1</SUP>state is obtained with a weak g=12 EPR signal, which increased in intensity at higher temperature and sharpened and intensified>10 fold in parallel- mode EPR. This allowed assignment of the g=12 EPR signal to an excited state of a non-Kramers spin system (presumably S=3). Previous Mössbauer and MCD spectroscopic measurements appeared to have been made with this redox state. A second oxidation by one electron (E <sub>m</sub> =+90 mV) led to the P <sup>OX2</SUP>redox state, which occurred as a spin mixture of S=1/2 and S=7/2 species. This redox state corresponded to the form obtained by the solid thionine oxidation procedure of Hagen and coworkers. Further oxidation of P <sup>OX2</SUP>redox state caused destruction of the iron-sulfur clusters and concommitant formation of S=9/2 and other high spin EPR signals.<p>During the efforts to obtain highly-purified Fe-hydrogenase and prismane protein from <em>Desulfovibrio vulgaris</em> (Hildenborough) a black protein with an ultraviolet-visible spectrum reminiscent of rubredoxin-like iron-sulfur centers was obtained. Subsequent biochemical and EPR spectroscopic characterization (Chapter 9) indicated that this protein was similar but not identical to the protein rubrerythrin isolated by Moura and coworkers. The new protein was called nigerythrin due to its black color and hemerythrin-like EPR signal. Although rubrerythrin was originally reported to contain two rubredoxin-like and one dinuclear iron center, metal analyses and spin quantitation revealed that rubrerythrin and nigerythrin each contain two rubredoxin-like and two dinuclear iron centers per homodimer. The three redox transitions in both proteins had midpoint potentials higher than +200 mV. This suggested that both proteins have a non-redox role with all six iron ions in the ferrous state.<p>In Chapter 10 a literature survey of non-integer and integer high spin systems in ironsulfur proteins is presented. In contrast to the well-documented occurrence of S=3/2 and S=2 spin states in [areas] <sup>1+</SUP>and [3Fe-4S] <sup>0</SUP>, respectively, the characterization of other, unusual iron-sulfur clusters with high spin states has not yet reached full maturity. The recent crystal structure of the MoFe protein of <em>Azotobacter vinelandii,</em> in which the FeMoco and P-clusters appeared to be larger clusters shows that the correlation between high spin states with structures other than the four basic iron-sulfur clusters indeed holds. The diversity of redox and spin states as observed for the prismane protein, desulfoviridin, carbonmonoxide dehydrogenase and Fe-hydrogenase indicates that besides the FeMoco and P-clusters other larger iron-sulfur clusters are present in biological systems.
Physiological roles and metabolism of fungal aryl alcohols
Jong, E. de - \ 1993
Agricultural University. Promotor(en): J.A.M. de Bont, co-promotor(en): M.M.G.R. Bol; J.A. Field. - S.l. : De Jong - ISBN 9789054851943 - 224
enzymen - bosbouw - pulpbereiding - biodegradatie - oxidoreductasen - peroxidasen - katalase - basidiomycotina - scheurvorming - decompositie - degradatie - lignine - chemische verbindingen - enzymes - forestry - pulping - biodegradation - oxidoreductases - peroxidases - catalase - cracking - decomposition - degradation - lignin - chemical compounds
<p>The major structural elements of wood and other vascular tissues are cellulose, hemicellulose and generally 20-30% lignin. Lignin gives the plant strength, it serves as a barrier against microbial attack and it acts as a water impermeable seal across cell walls of the xylem tissue. However, the presence of lignin has practical drawbacks for some of the applications of lignocellulosic materials. First, lignin has to be removed for the production of high quality pulps. Second, lignin reduces the digestibility of lignocellulosic materials. High quality pulps can be produced with chemical methods, however the abundant use of chemicals and energy, and the formation of an enormous waste stream has led scientists to investigate the possibilities of biodelignification. White-rot fungi give the most rapid and extensive degradation and have become subject of intensive research. Results obtained with the model organism <em>Phanerochaete chrysosporium</em> and other strains have revealed that lignin biodegradation is an extracellular, oxidative and non-specific process. This unique biodegradative potential has been considered for broader applications such as waste water treatment and the degradation of xenobiotic compounds. The research described in this thesis concentrates on the function of aryl alcohols in fungal physiology.<p><strong>Aryl alcohols In the physiology of white-rot fungi.</strong> White-rot fungi have a versatile machinery of enzymes, including peroxidases and oxidases, which work in harmony with secondary aryl alcohol metabolites to degrade the recalcitrant, aromatic biopolymer lignin. In chapter 2 literature concerning the important physiological roles of aryl (veratryl, anisyl and chlorinated anisyl) alcohols in the ligninolytic enzyme system has been reviewed. Their functions include stabilization of lignin peroxidase, charge-transfer reactions and as substrate for oxidases generating extracellular H <sub>2</sub> O <sub>2</sub> .<p>The experimental research described in this thesis was initiated to evaluate the possibilities of white-rot fungi in the biopulping of hemp stem wood. Sixty-seven basidiomycetes were isolated and screened for high peroxidative activity (chapter 3). Several of the new isolates were promising manganese peroxidase-producing white-rot fungi. Enzyme assays indicated that for the production of H <sub>2</sub> O <sub>2</sub> either extracellular glyoxal or aryl alcohol oxidase were present. In contrast, lignin peroxidase was only detected in <em>P. chrysosporium</em> , despite attempts to induce this enzyme in other strains with oxygen and oxygen/veratryl alcohol additions. A highly significant correlation was found between two ligninolytic indicators: ethene formation from α-keto-γ- methylthiolbutyric acid and the decolorization of a polymeric dye, Poly R-478. Three of the new isolates had significantly higher Poly R decolorizing activities compared to <em>P. chrysosporium</em> .<p>One of the best Poly R decolorizing strains, <em>Bjerkandera</em> sp. BOS55 was selected for further characterization. A novel enzyme activity (manganese independent peroxidase) was detected in the extracellular fluid of <em>Bjerkandera</em> sp. BOS55 (chapter 4). The purified enzyme could oxidize several compounds like phenol red, 2,6-dimethoxyphenol (DMP), Poly R-478, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and guaiacol with H <sub>2</sub> O <sub>2</sub> as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H <sub>2</sub> O <sub>2</sub> . <em>Bjerkandera</em> sp. BOS55 also produced de novo several aromatic metabolites. Besides veratryl alcohol and veratraldehyde, compounds which are known to be involved in the ligninolytic system of several other white-rot fungi (chapter 2), other metabolites were formed. These included anisaldehyde, 3-chloro-anisaldehyde, 3,5-dichloro-anisaldehyde and small amounts of the corresponding anisyl, 3-chloro-anisyl and 3,5-dichloro-anisyl alcohol (chapters 5 and 6). This was the first report of <em>de novo</em> biosynthesis of simple chlorinated aromatic compounds by a white-rot fungus. These unexpected findings led us investigate the physiological role(s) of the <em>de</em><em>novo</em> biosynthesized chlorinated anisyl alcohols (chapter 6). All metabolites were produced simultaneously with the extracellular ligninolytic enzymes. The monoand dichlorinated anisyl alcohols appeared to be excellent substrates for the extracellular aryl alcohol oxidases. The formed aldehydes were readily recycled via reduction by washed fungal mycelium, thus creating an extracellular H <sub>2</sub> O <sub>2</sub> production system regulated by intracellular enzymes. Lignin peroxidase does not oxidize the chlorinated anisyl alcohols both in the absence and in the presence of veratryl alcohol. It was therefore concluded that the chlorinated anisyl alcohols are well protected against the fungus's own aggressive ligninolytic enzymes. The relative amounts of veratryl alcohol and the chlorinated anisyl alcohols differ significantly depending on the growth conditions, indicating that the production of veratryl alcohol and the (chlorinated) anisyl metabolites are independently regulated.<p>It was concluded that the chlorinated anisyl metabolites, biosynthesized by the white-rot fungus <em>Bjerkandera</em> sp. BOS55, are purposeful for ecologically significant processes such as lignin degradation. These results made us speculate if a significant biogenesis of chlorinated aromatics by fungi occurs in natural environments (chapter 7). Many common wood- and forest litter-degrading fungi were indeed detected that produced chlorinated anisyl metabolites (CAM). These compounds, which are structurally related to xenobiotic chloroaromatics, were present in the environment and occur at high concentrations of approximately 75 mg CAM kg <sup>-1</SUP>wood or litter. The ubiquity among common fungi to produce large amounts of chlorinated aromatic compounds in the environment leads to the conclusion that these kind of compounds can no longer be considered to originate from anthropogenic sources only.<p><strong>Degradation of aryl alcohols by fungi.</strong> In chapter 2 the anabolic and catabolic routes of aryl alcohols by white-rot fungi has been reviewed. These fungi can not use veratryl alcohol as sole source of carbon and energy. However, several bacteria, yeasts and fungi were selectively isolated from paper mill waste water that grew on veratryl alcohol (chapter 8). <em>Penicillium</em><em>simplicissimum</em> was selected for the characterization of the veratryl alcohol degradation route. <em>P. simplicissimum</em> oxidized veratryl alcohol via a NAD(P) <sup>+</SUP>-dependent veratryl alcohol dehydrogenase to veratraldehyde which was further oxidized to veratric acid in a NAD(P) <sup>+</SUP>-dependent reaction. Veratric acid-grown cells contained NAD(P)H-dependent <em>O</em> -demethylase activity for veratrate, vanillate and isovanillate. Ring-cleavage of protocatechuate was by a protocatechuate 3,4-dioxygenase. An interesting aspect of <em>P. simplicissimum</em> is the production of vanillyl alcohol oxidase with covalently bound FAD (chapter 9). The intracellular enzyme was purified 32-fold. SDS-PAGE of the purified enzyme revealed a single fluorescent band of 65 Kda. Gel filtration and sedimentation-velocity experiments indicated that the purified enzyme exists in solution as an octamer, containing 1 molecule flavin/subunit. The covalently bound prosthetic group of the enzyme was identified as 8α-(N <sup>3</SUP>-histidyl)FAD from pH dependent fluorescence quenching (p <em>K</em><sub>a</sub> = 4.85) and no decrease in fluorescence upon reduction with sodium borohydride. The enzyme showed a narrow and rather peculiar substrate specificity. In addition to vanillyl alcohol and 4-hydroxybenzyl alcohol, eugenol and chavicol are substrates for the enzyme (chapter 10). The formed products, coniferyl and coumaryl alcohol are the natural precursors of lignin in plants. This reaction has a potential application to produce coniferyl alcohol and subsequent synthetic lignin (DHP) from the inexpensive precursor eugenol.
Molecular studies on iron-sulfur proteins in Desulfovibrio
Stokkermans, J. - \ 1993
Agricultural University. Promotor(en): C. Veeger; W.M.A.M. van Dongen. - S.l. : Stokkermans - 125
thiobacillus - sulfaat reducerende bacteriën - microbiële afbraak - microbiologie - zwavel - kringlopen - oxidoreductasen - polypeptiden - eiwitten - aminozuren - aminozuursequenties - sulfate reducing bacteria - microbial degradation - microbiology - sulfur - cycling - oxidoreductases - polypeptides - proteins - amino acids - amino acid sequences
<p><strong><em>Desulfovibrio vulgaris</em> (Hildenborough)</strong> . The organism described in this thesis, is an anaerobic gram-negative sulfate reducing bacterium (SRB). Its natural environments are the anaerobic sediments in lower levels of lakes and pools. This habitat is rich in sulfate that is used as terminal electron-acceptor by the organism and by performing this, <em>D. vulgaris</em> contributes to the important sulfur-cycle in nature. <em>D. vulgaris</em> can both utilize lactate (by anaerobic oxidation) and molecular hydrogen as energy source. The oxidation of lactate to acetate and C0 <sub>2</sub> occurs in the cytoplasm or at the cytoplasmic membrane and results in the production of ATP and the release of protons and electrons. When <em>D. vulgaris</em> uses molecular hydrogen as substrate, the oxidation of the hydrogen occurs at the periplasmic side of the inner membrane. This creates a proton motive force that drives ATP synthesis.<p><strong>Periplasmic Fe-hydrogenase</strong> . Molecular hydrogen has been shown to play an important role in both energy-evolving systems described above. So far, three hydrogenases, catalyzing the reversible H <sub>2</sub> oxidation and reduction of protons have been identified in <em>D. vulgaris</em> (Hildenborough). The precise physiological function of each of these hydrogenases remains unclear. Two of these enzymes are localized in the cytoplasmic membrane and contain nickel in addition to iron-sulfur clusters as cofactor. The third enzyme contains only iron-sulfur clusters as cofactors and resides in the periplasmic space of the bacterium. This enzyme exhibits one of the highest catalytic activities ever described for hydrogenases. It occurs as a heterodimer that is composed of a large cc subunit (46 kDa) and a small 0 subunit (10 kDa). Only the small subunit is translated as a precursor (13 kDa) with a cleavable signal sequence for export that is probably involved in the export of both hydrogenase subunits across the cytoplasmic membrane. The catalytically active enzyme contains three iron-sulfur clusters as cofactors. Two of them are typical ferredoxin-like [4Fe-4S] clusters (F-clusters) involved in electron transport. The third (H-cluster) cluster contains six iron and sulfide ions coordinated in an unknown structure and is part of the catalytic center of the enzyme.<p>Ile gene encoding the Fe-hydrogenase was the first hydrogenase gene that was isolated and expressed in <em>E. coli</em> . From these expression studies it became apparent that only very small amounts of αand βsubunits were assembled into an αβdimer and transported across the membrane. Also the iron-sulfur cluster incorporation was incomplete in the recombinant enzyme. The enzyme contained sub-stoichiometric amounts of F-clusters, while the H-cluster was not incorporated at all. These results indicated that the assembly and export of hydrogenase generating a catalytically active enzyme, are not spontaneously occurring processes, but involve specific helper components, as has been shown for other enzymes with redox-active metal clusters (reviewed in <strong>Chapter 1</strong> ).<p><strong>Studies regarding the biosynthesis of Fe-hydrogenase: the <em>hydC</em> gene</strong> . As genes serving a single pathway are often clustered in the genome, the identification of genes encoding these additional activating components, was started by the isolation of large DNA fragments surrounding the structural hydrogenase genes. Surprisingly, one of the large isolated DNA fragments contained a gene, <em>hydC</em> ( <strong>Chapter 2</strong> ) with homology in primary structure to the αand βsubunits of the Fe-hydrogenase.<br/>HydC has a high degree of similarity with both the αsubunit of the Fe-hydrogenase (in its central part) and with the βsubunit, minus the leader peptide (in its C-terminal part). Analogous to the FeMo co-factor insertion in nitrogenase component 1 which involves genes ( <em>nifEN</em> ) <em></em> with high similarities to the structural subunits, it was speculated that the <em>hydC</em> gene might code for a helper protein that is involved in the processing of the hydrogenase. 'Me primary structure of <em>hydC</em> contains a N-terminus with no homology with one of the hydrogenase subunits. Subsequently, it was found that this N-terminal segment has homology with mitochondrial NADH-ubiquinone reductase, with subunits of a NAD+-reducing NiFe-hydrogenase from <em>Alcaligenes eutrophus</em> and with the Fe-hydrogenase I from <em>Clostridium pasteurianum</em> ( <strong>Chapter 3</strong> ). On the basis of what is known about iron-sulfur cluster contents of these three enzymes and the conservation of cysteine motifs in these proteins, it was suggested that these motifs coordinate [2Fe-2S] clusters. Unfortunately, the HydC protein could not be purified from <em>D. vulgaris,</em> because no growth conditions were found resulting in a sufficient production of HydC protein. This hampered a further<br/>biochemical and spectroscopical characterization of the protein. On the other hand, the high degree of homology with the <em>C. pasteurianum</em> Fe-hydrogenase, strongly suggests that HydC is a second alternative Fe-hydrogenase and not a helper protein involved in the processing of Fe-hydrogenase.<p>Numerous attempts have been made to exchange the genes for the subunits of the Fe- hydrogenase and the <em>hydC</em> gene in the <em>D. vulgaris</em> genome with inactivated, interrupted copies of the genes. This type of marker exchange experiments would also be very useful for the identification of genes involved in biosynthesis of hydrogenase. One of the requirements for marker exchange is a system for the introduction of plasmids into <em>Desulfovibrio.</em> Such a plasmid transfer system has been developed, but subsequent experiments to apply it for marker exchange have been unsuccessful.<p><strong>The prismane protein</strong> . The inability to design a system for marker-exchange mutagenesis in <em>Desulfovibrio</em> blocked further study of the biosynthesis of the Fe-hydrogenase Therefore, investigations on another protein from <em>D. vulgaris,</em> the prismane protein, were started that are described in the second part of this thesis. As mentioned earlier, some indications were obtained that the H-cluster of Fe-hydrogenase is a [6Fe-6S] cluster.<p>Stronger indications for the existence of such "supercluster" were obtained by Hagen and Pierik in our department for another iron-sulfur containing protein from <em>D. vulgaris,</em> the prismane protein. They isolated a protein containing six irons and sulfide ions coordinated in only one [6Fe-6S] cluster. The putative [6Fe-6S] prismane cluster occurs in four different redox- states: the three-electron reduced state [6Fe-6S] <sup>3+</SUP>(S=1/2), [6Fe-6S] <sup>4+</SUP>(S = even), [6Fe-6S] <sup>5+</SUP>(S = 1/2 and S = 9/2) and the fully oxidized [6Fe-6S] <sup>6+</SUP>(S=0) that shows no EPR spectrum. <strong>Chapter 4</strong> and <strong>5</strong> describe the isolation of the genes for the prismane proteins from <em>D. vulgaris</em> (Hildenborough) and <em>D. desulfuricans</em> (ATCC 27774) and the determination of the amino acid sequence. Both proteins are highly conserved (66% identical residues), except for a 100 residues segment (residue 50-150). Besides this, both proteins contain typical cysteine motifs at the N-terminus. These motifs have also been found in the sequence of the a subunit of CO dehydrogenase from <em>Methanothrix soehngenii</em> and, in a slightly modified form, in that of CO dehydrogenase from <em>Clostridium thermoaceticum.</em> Also <em></em> for the CO dehydrogenase from <em>M. soehngenii</em> a supercluster has been proposed. Therefore, it is tempting to speculate about the involvement of this motif in the ligation of the [6Fe-6S] prismane cluster.<p>Prismane protein is produced only in small amounts in <em>D. vulgaris.</em> Since large amounts of purified prismane protein are required for X-ray crystallography and Mössbauer studies, efforts were made for overproduction of the protein ( <strong>Chapter 6</strong> ). In a first attempt, the protein was overproduced in <em>E. coli.</em> In this host, a high production of prismane protein was obtained, but no iron-sulfur cluster was incorporated into the protein. The overproduced protein occurred as large insoluble protein-complexes. A second attempt for the overproduction of prismane protein was performed in <em>D. vulgaris</em> by using the aforementioned cloning system. <em>A</em> 25-fold overproduction of prismane protein was obtained by the introduction of extra copies of the gene encoding the prismane protein on a stable plasmid. Biochemical and spectroscopic properties of the protein overproduced in <em>D. vulgaris</em> were shown to be identical to wild-type prismane with one exception: in the as-isolated, oneelectron-reduced state the protein shows EPR signals belonging to a second (S=1/2), spin system that was not observed in the wild-type protein. These additional signals were also described for the wild-type prismane protein purified from <em>D. desulfuricans</em> by Moura and co-workers in Portugal. EPR signals belonging to this second (S=1/2), spin system disappear upon reduction/re-oxidation of the overproduced prismane protein, indicating that this spin system represents a different magnetic form of the [6Fe-6S] cluster. There are no indications for a second cluster as proposed by Moura et al. Determination of the three dimensional structure by X-ray crystallography and further Mössbauer spectroscopy of the overproduced prismane protein are subject for further study in our department and will ultimately lead to insight into the structure of this novel iron-sulfur cofactor.
Studies on lipoamide dehydrogenase
Benen, J.A.E. - \ 1992
Agricultural University. Promotor(en): C. Veeger; A. de Kok. - S.l. : Benen - 132
oxidoreductasen - lipiden - moleculaire genetica - enzymen - kinetica - oxidoreductases - lipids - molecular genetics - enzymes - kinetics
At the onset of the investigations described in this thesis progress was being made on the elucidation of the crystal structure of the <em>Azotobacter</em><em>vinelandii</em> lipoamide dehydrogenase. Also the gene encoding this enzyme was cloned in our laboratory. By this, a firm basis was laid to start site directed mutagenesis studies aimed at deepening the insight in the reaction mechanism of lipoamide dehydrogenase. At the start of this work no site directed mutated mutated lipoamide dehydrogenases were reported though for the <em>E. coli</em> enzyme some mutated enzymes were announced.<p>The first goal was to assess the function(s) of the active site base, histidine. Therefore the histidine was replaced by other residues and a glutamate, held responsible for affecting the pK <sub>a</sub> of the histidine was mutagenised. It was expected that<br/>the mutations would alter the reaction rates of the different reaction intermediates and that these intermediates could be identified and studied.<p>Chapter 2 describes the steady state spectral properties and in Chapter 3 the steady state and rapid reaction kinetics and some rapid reaction spectral properties of the wildtype and mutated enzymes are presented.<p>Since modeling studies suggested that Tyr16 was involved in substrate binding, this residue was replaced with other residues in order to stabilize the binding of the substrate, lipS <sub>2</sub> , and hence to be able to grow crystals of the enzyme with the substrate bound. The results obtained with these mutated enzymes are presented in Chapter 4.<p>Preliminary experiments with <em>A. vinelandii, E. coli</em> and <em>P. fluorescens</em> lipoamide dehydrogenase showed strong reactivity of the latter enzyme with antibodies raised against <em>A. vinelandii</em> enzyme while no cross reactivity was observed with the <em>E. coli</em> enzyme. This indicates that high sequence homology is present between the <em>A. vinelandii</em> and the <em>P. fluorescens</em> enzymes and that the <em>P.</em><em>fluorescens</em> enzyme constitutes a natural 'mutated' effective lipoamide dehydrogenase. Cloning and sequence analysis of this gene, described in Chapter 5 opens the way to site directed mutagenesis. In Chapter 6 a characterization of the <em>P. fluorescens</em> wild-type enzyme is presented.
Studies of the quantitative structure-activity relationship of the inhibition of xanthine oxidase by azaheterocyclic compounds
Naeff, H.S.D. - \ 1990
Agricultural University. Promotor(en): H.C. van der Plas. - S.l. : Naeff - 87
structuuractiviteitsrelaties - xanthine - oxidoreductasen - structure activity relationships - oxidoreductases
<p>This thesis contains the results of a QSAR analysis of the interaction of bovine milk xanthine oxidase with two azaheterocyclic compounds, namely the 6-arylpteridin- 4-ones and the 8-arylhypoxanthines. Xanthine oxidase has active sites for various substrates. The studies done for this thesis were of the active site connected to the molybdenum cofactor.<p>Chapter 2 contains a description of how the 6-arylpteridin-4-ones and the 8-arylhypoxanthines were prepared. To synthesize the congeneric series of pteridines, the Gabriel-Isay method was used. This method, in which 4,5-diamino-6-hydroxypyrimidin reacts with an arylglyoxal, invariably led to contamination of the reaction product with isomeric 7-arylpteridin-4-ones. Multiple recrystallization from a DMSO-water solution minimizes contamination of the product to less than 5%. To synthesize the 8-arylhypoxanthines, benzoic acid derivatives were used instead of arylglyoxal derivatives.<p>The 6-arylpteridin-4-ones are good inhibitors of both free and immobilized xanthine oxidase (Chapter 3). To study the inhibitory properties of these compounds as expressed by their K <sub>i</sub> values, a QSAR equation was calculated. The equation shows that the electronic character of the 6-arylpteridin-4-one substituents does not influence the inhibition of xanthine oxidase; it is governed only by steric factors. The QSAR equation also shows that the effectiveness of an inhibitor is reduced by large spherical substituents like <em>t</em> -butyl and enhanced by rod-shaped substituents like <em>n</em> -butyl. For immobilized xanthine oxidase, an additional factor, namely the hydrophobic parameter π, is essential for the equation. The explanation for this lies in the hydrophilic character of the Sepharose matrix that is used to immobilize the enzyme.<p>The 8-arylhypoxanthines were synthesized and tested for their effectiveness as substrates of xanthine oxidase (Chapter 4). During synthesis, the 8-arylhypoxanthines are converted into the corresponding 8-arylxanthines. To characterize the product, the 8-phenylhypoxanthine was incubated with immobilized xanthine oxidase. The product of the enzymatic reaction was then isolated and characterized as 8- phenylxanthine by 13C-NMR and IR spectroscopy. The oxidation of these compounds is so slow that a detailed study was done only for the upsubstituted compound, the <strong>p</strong> -methyl, and the <strong>p</strong> -bromo substituted 8-arylliypoxanthines. At a high pH level, V <sub>max</sub> is considerably higher than at a low level and K <sub>m</sub> remains essentially the same. V <sub>max</sub> and K <sub>m</sub> are both higher at high temperatures.<p>Chapter 5 contains the results of experiments on the inhibition of free xanthine oxidase by the 8-arylhypoxanthines. At very low concentrations, the 8-arylhypoxanthines are effective inhibitors of xanthine oxidase. Consequently, the enzymatic oxidation of the 8-arylhypoxanthines (described in Chapter 4) heavily influences their resulting effective concentration. This makes it so difficult to measure the K <sub>i</sub> parameters accurately that only the 150 values are given here. The 8-arylhypoxanthines inhibit xanthine oxidase at equimolar concentrations, and the 8-arylxanthines have much higher 150 values. Therefore, because the 150 values of the 8-arylxanthines are similar to those of the 6-arylpteridin-4-ones, the 8-arylhypoxanthines are just as effective at inhibiting xanthine oxidase as the 6-arylpteridin-4-ones.<p>Chapter 6 contains a comparison of the inhibitory properties of the 6- arylpteridin-4-ones and the 8-arylhypoxanthines with those of several other azaheterocyclic compounds. The flexibility of the active site of xanthine oxidase is confirmed. The site, which contains the molybdenum co-factor, can accommodate compounds of different sizes. Proof of this is found in the inhibition of xanthine oxidase by a small compound like allopurinol, by larger compounds like 6-arylpteridin-4-one or 8-arylhypoxanthine, and by <em>lin</em> -naphthoxanthine, which is twice as large as allopurinol.<p>With these congeneric compound series, one can also study the "wall" of an enzyme's active site. The interaction of the substituents of the 6-arylpteridin-4-ones and the 8-aryl(hypo-)xanthines with the wall of xanthine oxidase's active site seems to be governed only by steric factors. This is because electronic parameters like σdo not appear in the QSAR equations. The type I and type II binding models explain the large differences between the 150 values of the 8-arylhypoxanthines and those of the 8-arylxanthines. Although these compounds are structurally similar, their aryl substituent interacts with different parts of the active site.
Structural studies on dihydrolipoyl transacetylase : the core component of the pyruvate dehydrogenase complex of Azotobacter vinelandii
Hanemaaijer, J.R.O. - \ 1988
Agricultural University. Promotor(en): C. Veeger, co-promotor(en): A. de Kok. - S.l. : Hanemaaijer - 119
azotobacter - koolhydraatmetabolisme - oxidoreductasen - carbohydrate metabolism - oxidoreductases
The studies described in this thesis deal with the structure of the <u>Azotobacter</u><u>vinelandii</u> dihydrolipoyl transacetylase, the core component (E <sub><font size="-1">2</font></sub> ) of the pyruvate dehydrogenase complex. in all organisms the pyruvate dehydrogenase complex is closely related to the 2-oxoglutarate dehydrogenase complex and, if present, the branched-chain 2-oxoacid dehydrogenase complex. These enzyme complexes are large multimeric structures. The smallest known is the pyruvate dehydrogenase complex from <u>A.vinelandii</u> , Upon resolution of the other components, the tetrameric core component of this complex aggregates to a welldefined multimeric structure, resembling the structure from the large complexes from other organisms.. Therefore, it seems likely that the <u>A.vinelandii</u> complex could represent the model for the building unit of the large complexes from other organisms. Since the core component (E <sub><font size="-1">2</font></sub> ) carries all the information concerning the quaternary structure of the complex, we focussed our attention on this intriguing enzyme.<p>The domain structure of E <sub><font size="-1">2</font></sub> has been examined by limited proteolysis of E <sub><font size="-1">2</font></sub> , as described in chapter 2. After limited proteolysis with trypsin two stable domains were obtained. The lipoyl domain carries the lipoyl groups which are concerned with the transport of the substrates between the active sites of the different components. The catalytic domain possesses the transacetylase active site and the E <sub><font size="-1">2</font></sub> -intersubunit binding sites, responsible for the quaternary structure of E <sub><font size="-1">2</font></sub> . The binding sites for the E <sub><font size="-1">1</font></sub> and E <sub><font size="-1">3</font></sub> components are lost during proteolysis.<p>The cloning and sequencing of the gene encoding dihydrolipoyl transacetylase have been described in chapter 3. The gene, located downstream of the gene encoding the PDC E <sub><font size="-1">1</font></sub> component, does not possess an own promoter, but is probably regulated by the E <sub><font size="-1">1</font></sub> -promoter. The gene possesses a strong terminating sequence. Downstream the gene encoding E <sub><font size="-1">2</font></sub> no open reading frame, that codes for the E <sub><font size="-1">3</font></sub> component, has been identified, as has been found in <u>E.coli</u> . The primary structure of E <sub><font size="-1">2</font></sub> , derived from the DNA sequence, is homologous to that of E <sub><font size="-1">2</font></sub> from <u>E.coli</u> . The lipoyl domain, located at the N-terminus, is built from three repeating sequences, separated by regions which are very rich in alanine and proline residues. The catalytic domain, located at the C-terminus, comprises the transacetylase active site and the E <sub><font size="-1">2</font></sub> intersubunit binding sites. The region, located between the lipoyl and the catalytic domain contains many charged amino acid residues and is thought to possess the E <sub><font size="-1">1</font></sub> and E <sub><font size="-1">3</font></sub> binding sites. The expression of the gene encoding E <sub><font size="-1">2</font></sub> , located on plasmid pRA282 and cloned in <u>E.coli</u> , has been described in chapter 4. A high production of E <sub><font size="-1">2</font></sub> was obtained. The production raised dramatically when the cells were in the stationery phase of the growth-cycle. The percentage active E <sub><font size="-1">2</font></sub> varied strongly per culture. The inactivation was found to be caused by formation of intramolecular or intermolecular S-S-bridges, resulting in incorrect folding of the catalytic domain. An activation and an isolation procedure have been described.<p>Mobility of the repeating units within the lipoyl domain has been studied using time-resolved fluorescence, as described in chapter 5. It has been shown that the repeats show no independent rotational mobility, but rotate as one unit, serving the active sites of the different components.<p>Internal mobility within the lipoyl domain has been observed by <sup><font size="-2">1</font></SUP>H-NMR experiments, as described in chapter 6. Probably this internal mobility, that is ascribed to the alanine-proline rich region, does not result into an independent mobility of the three repeats. The catalytic domain, despite its compact structure, still possesses a certain amount of internal mobility. This can partly be ascribed to alanine and proline residues, probably the N-terminal region of the domain, which is rich in these residues. In the spectrum of E <sub><font size="-1">2</font></sub> sharp resonances have been observed that can be ascribed to mobility of the E <sub><font size="-1">1</font></sub> and E <sub><font size="-1">3</font></sub> binding domain. Such mobility has not been found after binding of E <sub><font size="-1">1</font></sub> and E <sub><font size="-1">3</font></sub> components, in the whole complex.<p>The molecular mass of the native catalytic domain and of the single polypeptide chain have been determined, and from this and light-scattering and crosslinking experiments it has been concluded that the large multimeric structure of the isolated catalytic domain (and of E <sub><font size="-1">2</font></sub> ) is built from 24 subunits in contrast to a 32-meric structure as proposed previously. A model has been presented for the quaternary structure of E <sub><font size="-1">2</font></sub> , in which it is assumed that the multimeric E <sub><font size="-1">2</font></sub> -core is built from six tetrameric morphological subunits, forming the lateral faces of the cubic 24-mer.<p>These tetrameric subunits represent the E2-core of the intact complex. Compared to other 2-oxoacid dehydrogenase complexes, the <u>A.vinelandii</u> PDC contains one additional binding site for E <sub><font size="-1">1</font></sub> per E <sub><font size="-1">2</font></sub> tetramer. It is assumed that this extra binding site becomes available during dissociation, resulting in the unique small PDC of <u>A.vinelandii</u> .<p><TT></TT>
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