Staff Publications

Staff Publications

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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.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    DHAP-dependent aldolases from (hyper)thermophiles: biochemistry and applications
    Falcicchio, P. ; Wolterink-van Loo, S. ; Franssen, M.C.R. ; Oost, J. van der - \ 2014
    Extremophiles 18 (2014)1. - ISSN 1431-0651 - p. 1 - 13.
    l-threonine aldolase - stereochemically complementary biocatalysts - ii fructose-1,6-bisphosphate aldolase - thermus-thermophilus hb8 - escherichia-coli - d-fructose-6-phosphate aldolase - directed evolution - crystal-structure - active-site - l-rhamnulose-1-phosp
    Generating new carbon-carbon (C-C) bonds in an enantioselective way is one of the big challenges in organic synthesis. Aldolases are a natural tool for stereoselective C-C bond formation in a green and sustainable way. This review will focus on thermophilic aldolases in general and on dihydroxyacetone phosphate-dependent aldolases in particular. Biochemical properties and applications for synthesis of rare sugars and carbohydrates will be discussed
    Fungal endopolygalacturonases are recognized as MAMPs by the Arabidopsis Receptor-Like Protein RBPG1
    Zhang, L. ; Kars, I. ; Essenstam, B. ; Liebrand, T.W.H. ; Wagemakers, L. ; Elberse, J. ; Tagkalaki, P. ; Tjoitang, D. ; Ackerveken, G. van den; Kan, J.A.L. van - \ 2014
    Plant Physiology 164 (2014)1. - ISSN 0032-0889 - p. 352 - 364.
    ethylene-inducing xylanase - agrobacterium-mediated transformation - innate immunity - nicotiana-benthamiana - necrotizing activity - enzymatic-activity - aspergillus-niger - plasma-membrane - plant immunity - active-site
    Plants perceive microbial invaders using pattern recognition receptors that recognize microbe-associated molecular patterns. In this study, we identified RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1), an Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like protein, AtRLP42, that recognizes fungal endopolygalacturonases (PGs) and acts as a novel microbe-associated molecular pattern receptor. RBPG1 recognizes several PGs from the plant pathogen Botrytis cinerea as well as one from the saprotroph Aspergillus niger. Infiltration of B. cinerea PGs into Arabidopsis accession Columbia induced a necrotic response, whereas accession Brno (Br-0) showed no symptoms. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the Columbia RBPG1 gene and showed that this gene is essential for the responsiveness of Arabidopsis to the PGs. Transformation of RBPG1 into accession Br-0 resulted in a gain of PG responsiveness. Transgenic Br-0 plants expressing RBPG1 were equally susceptible as the recipient Br-0 to the necrotroph B. cinerea and to the biotroph Hyaloperonospora arabidopsidis. Pretreating leaves of the transgenic plants with a PG resulted in increased resistance to H. arabidopsidis. Coimmunoprecipitation experiments demonstrated that RBPG1 and PG form a complex in Nicotiana benthamiana, which also involves the Arabidopsis leucine-rich repeat receptor-like protein SOBIR1 (for SUPPRESSOR OF BIR1). sobir1 mutant plants did not induce necrosis in response to PGs and were compromised in PG-induced resistance to H. arabidopsidis.
    Conformational landscapes of DNA polymerase I and mutator derivates establish fidelity checkpoints for nucleotide insertion
    Hohlbein, J.C. ; Aigrain, L. ; Craggs, T.D. ; Bermek, O. ; Potapova, O. ; Shoolizadeh, P. ; Grindley, N.D.F. ; Joyce, C.M. ; Kapanidis, A.N. - \ 2013
    Nature Communications 4 (2013). - ISSN 2041-1723 - 11 p.
    single-molecule fret - resonance energy-transfer - probability-distribution analysis - alternating-laser excitation - klenow fragment - escherichia-coli - active-site - photon distribution - mechanism - dynamics
    The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.
    The crystal structure of a hyperthermoactive exopolygalacturonase from Thermotoga maritima reveals a unique tetramer
    Pijning, T. ; Pouderoyen, G. van; Kluskens, L.D. ; Oost, J. van der; Dijkstra, B.W. - \ 2009
    FEBS Letters 583 (2009)22. - ISSN 0014-5793 - p. 3665 - 3670.
    active-site - endopolygalacturonase-i - aspergillus-aculeatus - sequence alignments - polygalacturonase - protein - features - crystallography - mutagenesis - family-28
    The exopolygalacturonase from Thermotoga maritima is the most thermoactive and thermostable pectinase known to date. Here we present its crystal structure at 2.05A resolution. High structural homology around the active site allowed us to propose a model for substrate binding, explaining the exo-cleavage activity and specificity for non-methylated saturated galacturonate at the non-reducing end. Furthermore, the structure reveals unique features that contribute to the formation of stable tetramers in solution. Such an oligomerization has not been observed before for polygalacturonases
    Molecular determinants of xenobiotic metabolism: QM/MM simulation of the conversion of 1-chloro-2,4-dinitrobenzene catalyzed by M1-1 glutathione S-transferase.
    Bowman, A.L. ; Ridder, L. ; Rietjens, I.M.C.M. ; Vervoort, J.J.M. ; Mulholland, A.J. - \ 2007
    Biochemistry 46 (2007)21. - ISSN 0006-2960 - p. 6353 - 6363.
    nucleophilic aromatic-substitution - potential free-energy - active-site - mu class - dynamics calculations - enzymatic-reactions - reaction-mechanisms - isoenzyme 3-3 - conjugation - evolution
    Modeling methods allow the identification and analysis of determinants of reactivity and specificity in enzymes. The reaction between glutathione and 1-chloro-2,4-dinitrobenzene (CDNB) is widely used as a standard activity assay for glutathione S-transferases (GSTs). It is important to understand the causes of differences between catalytic GST isoenzymes and the effects of mutations and genetic polymorphisms. Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations have been performed here to investigate the addition of the glutathione anion to CDNB in the wild-type M1-1 GST isoenzyme from rat and in three single point mutant (Tyr6Phe, Tyr115Phe, and Met108Ala) M1-1 GST enzymes. We have developed a specifically parameterized QM/MM method (AM1-SRP/CHARMM22) to model this reaction by fitting to experimental heats of formation and ionization potentials. Free energy profiles were obtained from molecular dynamics simulations of the reaction using umbrella sampling and weighted histogram analysis techniques. The reaction in solution has also been simulated and is compared to the enzymatic reaction. The free energies are in excellent agreement with experimental results. Overall the results of the present study show that QM/MM reaction pathway analysis provides detailed insight into the chemistry of GST and can be used to obtain mechanistic insight into the effects of specific mutations on this catalytic process.
    Joosten, V. ; Berkel, W.J.H. van - \ 2007
    Current Opinion in Chemical Biology 11 (2007)2. - ISSN 1367-5931 - p. 195 - 202.
    baeyer-villiger monooxygenase - p-hydroxybenzoate hydroxylase - crystal-structure - active-site - heterologous expression - histone demethylase - substrate-binding - escherichia-coli - structural basis - oxidase
    Flavoenzymes are colourful oxidoreductases that catalyze a large variety of different types of reactions. Flavoenzymes have been extensively studied for their structural and mechanistic properties and are gaining momentum in industrial biocatalytic applications. Some of these enzymes catalyze the oxidative modification of protein substrates. New insights in oxidative flavoenzymes and in particular in novel family members point towards their potential application in the pharmaceutical, fine-chemical and food industries
    Deletion of PREPL, a gene encoding a putative serine oligopeptidase, in patients with hypotonia-cystinuria syndrome
    Jaeken, J. ; Martens, K. ; Francois, I. ; Eyskens, F. ; Lecointre, C. ; Derua, R. ; Meulemans, S. ; Slootstra, J.W. ; Waelkens, E. ; Zegher, F. de; Creemers, J.W.M. ; Matthijs, G. - \ 2006
    American Journal of Human Genetics 78 (2006)1. - ISSN 0002-9297 - p. 38 - 51.
    alpha/beta-hydrolase fold - host-cell invasion - prolyl oligopeptidase - trypanosoma-cruzi - peptide libraries - processing enzyme - active-site - endopeptidase - substrate - mutations
    In 11 patients with a recessive congenital disorder, which we refer to as ¿the hypotonia-cystinuria syndrome,¿ microdeletion of part of the SLC3A1 and PREPL genes on chromosome 2p21 was found. Patients present with generalized hypotonia at birth, nephrolithiasis, growth hormone deficiency, minor facial dysmorphism, and failure to thrive, followed by hyperphagia and rapid weight gain in late childhood. Since loss-of-function mutations in SLC3A1 are known to cause isolated cystinuria type I, and since the expression of the flanking genes, C2orf34 and PPM1B, was normal, the extended phenotype can be attributed to the deletion of PREPL. PREPL is localized in the cytosol and shows homology with prolyl endopeptidase and oligopeptidase B. Substitution of the predicted catalytic residues (Ser470, Asp556, and His601) by alanines resulted in loss of reactivity with a serine hydrolase-specific probe. In sharp contrast to prolyl oligopeptidase and oligopeptidase B, which require both aminoterminal and carboxyterminal sequences for activity, PREPL activity appears to depend only on the carboxyterminal domain. Taken together, these results suggest that PREPL is a novel oligopeptidase, with unique structural and functional characteristics, involved in hypotonia-cystinuria syndrome.
    Reduced proteolysis of secreted gelatin and Yps1-Mediated alpha-factor leader processing in a Pichia pastoris kex2 disruptant
    Werten, M.W.T. ; Wolf, F.A. de - \ 2005
    Applied and Environmental Microbiology 71 (2005)5. - ISSN 0099-2240 - p. 2310 - 2317.
    aspartic protease 3 - yapsin 1 yap3p - saccharomyces-cerevisiae - kex2 protease - pichia-pastoris - schizosaccharomyces-pombe - active-site - in-vivo - yeast - proteins
    Heterologous proteins secreted by yeast and fungal expression hosts are occasionally degraded at basic amino acids. We cloned Pichia pastoris homologs of the Saccharomyces cerevisiae basic residue-specific endoproteases Kex2 and Yps1 to evaluate their involvement in the degradation of a secreted mammalian gelatin. Disruption of the P. pastoris KEX2 gene prevented proteolysis of the foreign protein at specific monoarginylic sites. The S. cerevisiae -factor preproleader used to direct high-level gelatin secretion was correctly processed at its dibasic site in the absence of the prototypical proprotein convertase Kex2. Disruption of the YPS1 gene had no effect on gelatin degradation or processing of the -factor propeptide. When both the KEX2 and YPS1 genes were disrupted, correct precursor maturation no longer occurred. The different substrate specificities of both proteases and their mutual redundancy for propeptide processing indicate that P. pastoris kex2 and yps1 single-gene disruptants can be used for the -factor leader-directed secretion of heterologous proteins otherwise degraded at basic residues.
    Ligand-induced changes in the conformational dynamics of a bacterial cytotoxic endonuclease
    Bremer, E.T.J. van den; Keeble, A.H. ; Visser, A.J.W.G. ; Hoek, A. van; Kleanthous, C. ; Heck, A.J.R. ; Jiskoot, W. - \ 2004
    Biochemistry 43 (2004)14. - ISSN 0006-2960 - p. 4347 - 4355.
    protein-protein interactions - single-tryptophan mutants - n-h motif - colicin e9 - transition-metals - immunity proteins - escherichia-coli - dnase domain - active-site - complex
    Knowledge about the conformational dynamics of a protein is key to understanding its biochemical and biophysical properties. In the present work we investigated the dynamic properties of the enzymatic domain of DNase colicins via time-resolved fluorescence and anisotropy decay analysis in combination with steady-state acrylamide quenching experiments. The dynamic properties of the apoenzyme were compared to those of the E9 DNase ligated to the transition metal ion Zn2+ and the natural inhibitor Im9. We further investigated the contributions of each of the two tryptophans within the E9 DNase (Trp22 and Trp58) using two single-tryptophan mutants (E9 W22F and E9 W58F). Wild-type E9 DNase, E9 W22F, and E9 W58F, as well as Im9, showed multiple lifetime decays. The time-resolved and steady-state fluorescence results indicated that complexation of E9 DNase with Zn2+ induces compaction of the E9 DNase structure, accompanied by immobilization of Trp22 along with a reduced solvent accessibility for both tryptophans. Im9 binding resulted in immobilization of Trp22 along with a decrease in the longest lifetime component. In contrast, Trp58 experienced less restriction on complexation of E9 DNase with Im9 and showed an increase in the longest lifetime component. Furthermore, the results point out that the Im9-induced changes in the conformational dynamics of E9 DNase are predominant and occur independently of the Zn2+-induced conformational effects.
    Quenching of quercetin quinone/quinone methides by different thiolate scavengers: stability and reversibility of conjugate formation
    Awad, H.M. ; Boersma, M.G. ; Boeren, J.A. ; Bladeren, P.J. van; Vervoort, J.J.M. ; Rietjens, I.M.C.M. - \ 2003
    Chemical Research in Toxicology 16 (2003)7. - ISSN 0893-228X - p. 822 - 831.
    glutathione s-transferases - irreversible inhibition - lipid-peroxidation - mutagenic activity - quinone methide - active-site - o-quinones - oxidation - flavonoids - antioxidant
    Oxidation of flavonoids with a catechol structural motif in their B ring leads to formation of flavonoid quinone/quinone methides, which rapidly react with GSH to give reversible glutathionyl flavonoid adducts. Results of the present study demonstrate that as a thiol-scavenging agent for this reaction Cys is preferred over GSH and N-acetyleysteine. The preferential scavenging by Cys over GSH reported in the present study appeared not to provide a basis for detection of thiol-based flavonoid conjugates in biological systems. This is because physiological concentrations of GSH are substantially higher than those of Cys, which was shown to shift the balance of thiol conjugate formation in favor of glutathionyl adduct formation. Furthermore, the cysteinyl quercetin adducts, although not showing the reversible nature of the glutathionyl conjugates, appeared nevertheless to be unstable. Thus, as a biomarker for formation of reactive quercetin quinone/quinone methides in biological systems, detection of the glutathionyl conjugates or the N-acetyleysteinyl conjugates derived from them should still be the method of choice. At GSH levels that dominate the level of other cellular thiol groups, covalent addition of the quinone to other cellular thiol groups may be efficiently prevented. However, various tissues are known to contain higher levels of protein-bound sulfhydryl moieties than of nonprotein sulfhydryl groups, the latter consisting of especially GSH. Thus, the results of the present study indicate that in biological systems covalent addition of quercetin quinone methide to tissue protein sulfhydryl groups can be expected. The transient nature of these adducts, as shown for all three types of thiol quercetin adducts in the present study, will, however, also result in a transient nature of the protein-bound quercetin adducts to be expected. Because stability of the various thiol quercetin adducts appeared a matter of minutes to hours instead of days, this rapid transient nature of possible quercetin quinone methide adducts may also restrict the ultimate toxicity to be expected from the quercetin quinone/quinone methides.
    Inhibition of human glutathione S-transferase P1-1 by the flavonoid quercetin
    Zanden, J.J. van; Hamman, O. Ben; Iersel, M.L. van; Boeren, J.A. ; Cnubben, N.H.P. ; Bello, M. Lo; Vervoort, J.J.M. ; Bladeren, P.J. van; Rietjens, I.M.C.M. - \ 2003
    Chemico-Biological Interactions 145 (2003)2. - ISSN 0009-2797 - p. 139 - 148.
    site-directed mutagenesis - human placenta - quinone methide - ethacrynic-acid - active-site - pi - identification - consequences - inactivation - conjugation
    In the present study, the inhibition of human glutathione S-transferase P1-1 (GSTP1-1) by the flavonoid quercetin has been investigated. The results show a time- and concentration-dependent inhibition of GSTP1-1 by quercetin. GSTP1-1 activity is completely inhibited upon I h incubation with 100 muM quercetin or 2 h incubation with 25 muM quercetin, whereas 1 and 10 muM quercetin inhibit GSTP1-1 activity to a significant extent reaching a maximum of 25 and 42% inhibition respectively after 2 h. Co-incubation with tyrosinase greatly enhances the rate of inactivation, whereas co-incubation with ascorbic acid or glutathione prevents this inhibition. Addition of glutathione upon complete inactivation of GSTP1-1 partially restores the activity. Inhibition studies with the GSTP1-1 mutants C47S, C101S and the double mutant C47S/C101S showed that cysteine 47 is the key residue in the interaction between quercetin and GSTP1-1. HPLC and LGMS analysis of trypsin digested GSTP1-1 inhibited by quercetin did not show formation of a covalent bond between Cys 47 residue of the peptide fragment 45-54 and quercetin. It was demonstrated that the inability to detect the covalent quercetin-peptide adduct using LGMS is due to the reversible nature of the adduct-formation in combination with rapid and preferential dimerization of the peptide fragment once liberated from the protein. Nevertheless, the results of the present study indicate that quinone-type oxidation products of quercetin likely act as specific active site inhibitors of GSTP1-1 by binding to cysteine 47. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.
    Quantum Mechanical/Molecular Mechanical Free Energy Simulations of the Glutathione S-Transferase (M1-1) Reaction with Phenanthrene 9,10-Oxide
    Ridder, L. ; Rietjens, I.M.C.M. ; Vervoort, J.J.M. ; Mulholland, A.J. - \ 2002
    Journal of the American Chemical Society 124 (2002)33. - ISSN 0002-7863 - p. 9926 - 9936.
    direct dynamics calculations - active-site - catalytic mechanism - molecular-dynamics - hydrogen-bond - computer-simulation - citrate synthase - enzyme reaction - aromatic hydroxylation - reaction pathway
    Glutathione S-transferases (GSTs) play an important role in the detoxification of xenobiotics in mammals. They catalyze the conjugation of glutathione to a wide range of electrophilic compounds. Phenanthrene 9,10-oxide is a model substrate for GSTs, representing an important group of epoxide substrates. In the present study, combined quantum mechanical/molecular mechanical (QM/MM) simulations of the conjugation of glutathione to phenanthrene 9,10-oxide, catalyzed by the M1-1 isoenzyme from rat, have been carried out to obtain insight into details of the reaction mechanism and the role of solvent present in the highly solvent accessible active site. Reaction-specific AM1 parameters for sulfur have been developed to obtain an accurate modeling of the reaction, and QM/MM solvent interactions in the model have been calibrated. Free energy profiles for the formation of two diastereomeric products were obtained from molecular dynamics simulations of the enzyme, using umbrella sampling and weighted histogram analysis techniques. The barriers (20 kcal/mol) are in good agreement with the overall experimental rate constant and with the formation of equal amounts of the two diastereomeric products, as experimentally observed. Along the reaction pathway, desolvation of the thiolate sulfur of glutathione is observed, in agreement with solvent isotope experiments, as well as increased solvation of the epoxide oxygen of phenanthrene 9,10-oxide, illustrating an important stabilizing role for active site solvent molecules. Important active site interactions have been identified and analyzed. The catalytic effect of Tyr115 through a direct hydrogen bond with the epoxide oxygen of the substrate, which was proposed on the basis of the crystal structure of the (9S,10S) product complex, is supported by the simulations. The indirect interaction through a mediating water molecule, observed in the crystal structure of the (9R,10R) product complex, cannot be confirmed to play a role in the conjugation step. A selection of mutations is modeled. The Asn8Asp mutation, representing one of the differences between the M1-1 and M2-2 isoenzymes, is identified as a possible factor contributing to the difference in the ratio of product formation by these two isoenzymes. The QM/MM reaction pathway simulations provide new and detailed insight into the reaction mechanism of this important class of detoxifying enzymes and illustrate the potential of QM/MM modeling to complement experimental data on enzyme reaction mechanisms
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