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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    IdsA is the major geranylgeranyl pyrophosphate synthase involved in carotenogenesis in Corynebacterium glutamicum
    Heider, S.A.E. ; Peters-Wendisch, P. ; Beekwilder, M.J. ; Wendisch, V.F. - \ 2014
    FEBS Journal 281 (2014)21. - ISSN 1742-464X - p. 4906 - 4920.
    farnesyl-diphosphate synthase - site-directed mutagenesis - chain-length determination - cytochrome-bo operon - escherichia-coli - crystal-structure - mycobacterium-tuberculosis - isoprenoid biosynthesis - conserved aspartate - micrococcus-luteus
    Corynebacterium glutamicum, a yellow-pigmented soil bacterium that synthesizes the rare cyclic C50 carotenoid decaprenoxanthin and its glucosides, has been engineered for the production of various carotenoids. CrtE was assumed to be the major geranylgeranyl pyrophosphate (GGPP) synthase in carotenogenesis; however, deletion of crtE did not abrogate carotenoid synthesis. In silico analysis of the repertoire of prenyltransferases encoded by the C. glutamicum genome revealed two candidate GGPPS genes (idsA and ispB). The absence of pigmentation of an idsA deletion mutant and complementation experiments with a double deletion mutant lacking both idsA and crtE showed that IdsA is the major GGPPS of C. glutamicum and that crtE overexpression compensated for the lack of IdsA, whereas plasmid-borne overexpression of ispB did not. Purified His-tagged CrtE was active as a homodimer, whereas the active form of IdsA was homotetrameric. Both enzymes catalyzed prenyl transfer with isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate, geranyl pyrophosphate and farnesylphosphate (FPP) as substrates. IdsA showed the highest catalytic efficiency with dimethylallyl pyrophosphate and IPP, whereas the catalytic efficiency of CrtE was highest with geranyl pyrophosphate and IPP. Finally, application of prenyltransferase overexpression revealed that combined overexpression of idsA and the IPP isomerase gene idi in the absence of crtE led to the highest decaprenoxanthin titer reported to date.
    Flavin dependent monooxygenases
    Huijbers, M.M.E. ; Montersino, S. ; Westphal, A.H. ; Tischler, D. ; Berkel, W.J.H. van - \ 2014
    Archives of Biochemistry and Biophysics 544 (2014). - ISSN 0003-9861 - p. 2 - 17.
    flavoprotein tryptophan 2-monooxygenase - baeyer-villiger monooxygenases - site-directed mutagenesis - p-hydroxyphenylacetate 3-hydroxylase - para-hydroxybenzoate hydroxylase - sphingobium-xenophagum bayram - l-phenylalanine oxidase - l-lactate monooxygenase - rh
    Flavin-dependent monooxygenases catalyze a wide variety of chemo-, regio- and enantioselective oxygenation reactions. As such, they are involved in key biological processes ranging from catabolism, detoxification and biosynthesis, to light emission and axon guidance. Based on fold and function, flavin-dependent monooxygenases can be distributed into eight groups. Groups A and B comprise enzymes that rely on NAD(P)H as external electron donor. Groups C–F are two-protein systems, composed of a monooxygenase and a flavin reductase. Groups G and H comprise internal monooxygenases that reduce the flavin cofactor through substrate oxidation. Recently, many new flavin-dependent monooxygenases have been discovered. In addition to posing basic enzymological questions, these proteins attract attention of pharmaceutical and fine-chemical industries, given their importance as regio- and enantioselective biocatalysts. In this review we present an update of the classification of flavin-dependent monooxygenases and summarize the latest advances in our understanding of their catalytic and structural properties
    Crystal structure of endo-xylogalacturonan hydrolase from Aspergillus tubingensis
    Rozeboom, H.J. ; Beldman, G. ; Schols, H.A. ; Dijkstra, B.W. - \ 2013
    FEBS Journal 280 (2013)23. - ISSN 1742-464X - p. 6061 - 6069.
    site-directed mutagenesis - endopolygalacturonase ii - sequence alignments - features - polysaccharides - processivity - degradation - pectin - niger - polygalacturonase
    Endo-xylogalacturonan hydrolase is a member of glycoside hydrolase family 28 (GH28) that hydrolyzes the glycosidic bond between two ß-xylose-substituted galacturonic acid residues in pectin. Presented here is the X-ray crystal structure of the endo-xylogalacturonan hydrolase from Aspergillus tubingensis (XghA) at 1.75 Å resolution. The high degree of structural conservation in the active site and catalytic apparatus compared with polygalacturonases indicates that cleavage of the substrate proceeds in essentially the same way as found for the other GH28 enzymes. Molecular modeling of a xylosylated tri-galacturonate in the active site identified the amino acid residues involved in substrate binding. They border a substrate-binding cleft that is much wider than in other polygalacturonases, and can accommodate xylosylated substrates. The most extensive interactions appear to occur at subsite +2, in agreement with the enzyme kinetics results, which showed enhanced activity on substrates with a xylose attached to the galacturonic acid bound at subsite +2
    Thermal Stabilization of an Endoglucanase by Cyclization
    Lieshout, J.F.T. van; Gutierrez, O. ; Vroom, W. ; Planas, A. ; Vos, W.M. de; Oost, J. van der; Koutsopoulos, S. - \ 2012
    Applied Biochemistry and Biotechnology 167 (2012)7. - ISSN 0273-2289 - p. 2039 - 2053.
    site-directed mutagenesis - beta-glucosidase celb - bacillus-licheniformis - circular proteins - in-vivo - pyrococcus-furiosus - crystal-structure - pi-pfui - stability - intein
    An intein-driven protein splicing approach allowed for the covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the endo-ß-1,3-1,4-glucanase, LicA, from Bacillus licheniformis. Two circular variants, LicA-C1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, showed catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA (LicA-L1). The thermal stability of the circular variants was significantly increased compared to the linear form. Whereas the linear glucanase lost half of its activity after 3 min at 65 °C, the two circular variants have 6-fold (LicA-C1) and 16-fold (LicA-C2) increased half-life time of inactivation. In agreement with this, fluorescence spectroscopy and differential scanning calorimetry studies revealed that circular enzymes undergo structural changes at higher temperatures compared to that of the linear form. The effect of calcium on the conformational stability and function of the circular LicAs was also investigated, and we observed that the presence of calcium ions results in increased thermal stability. The impact of the length of the designed loops on thermal stability of the circular proteins is discussed, and it is suggested that cyclization may be an efficient strategy for the increased stability of proteins
    Proteomic analysis of the major birch allergen Bet v 1 predicts allergenicity for 15 birch species
    Schenk, M.F. ; Cordewener, J.H.G. ; America, A.H.P. ; Peters, J. ; Smulders, M.J.M. ; Gilissen, L.J.W.J. - \ 2011
    Journal of Proteomics 74 (2011)8. - ISSN 1874-3919 - p. 1290 - 1300.
    mutational epitope analysis - site-directed mutagenesis - pollen allergen - recombinant allergens - phylogenetic-relationships - cross-reactivity - apple cultivars - ige reactivity - potential use - bet-v-1
    Pollen of the European and Asian white birch (Betula pendula and B. platyphylla) causes hay fever in humans. The allergenic potency of other birch species is largely unknown. To identify birch trees with a reduced allergenicity, we assessed the immunochemical characteristics of 15 species and two hybrids, representing four subgenera within the genus Betula, while focusing on the major pollen allergen Bet v 1. Antigenic and allergenic profiles of pollen extracts from these species were evaluated by SDS-PAGE and Western blot using pooled sera of birch-allergic individuals. Tryptic digests of the Bet v 1 bands were analyzed by LC-MSE to determine the abundance of various Bet v 1 isoforms. Bet v 1 was the most abundant pollen protein across all birch species. LC-MSE confirmed that pollen of all species contained a mixture of multiple Bet v 1 isoforms. Considerable differences in Bet v 1 isoform composition exist between birch species. However, isoforms that are predicted to have a high IgE-reactivity prevailed in pollen of all species. Immunoblotting confirmed that all pollen extracts were similar in immune-reactivity, implying that pollen of all birch species is likely to evoke strong allergic reactions.
    Assessment of the pectin degrading enzyme network of Aspergillus niger by functional genomics
    Martens-Uzunova, E.S. ; Schaap, P.J. - \ 2009
    Fungal Genetics and Biology 46 (2009)Suppl. 1. - ISSN 1087-1845 - p. S170 - S179.
    site-directed mutagenesis - cell wall polysaccharides - endopolygalacturonase-ii - glycoside hydrolases - encoding gene - lyase-a - expression - degradation - identification - residues
    The saprobic fungus Aspergillus niger is an efficient producer of a suite of extracellular enzymes involved in carbohydrate modification and degradation. Genome mining has resulted in the prediction of at least 39 genes encoding enzymes involved in the depolymerisation of the backbone of pectin. Additional genes,encoding enzymatic activities required for the degradation of the arabinan and arabinogalactan sidechains were predicted as well. DNA microarray analysis was used to study the condition-dependent expression of these genes, and to generate insights in possible synergistic interactions between the individual members of the pectin degrading enzyme network. For this purpose, A. niger was grown on sugarbeet pectin and on galacturonic acid, rhamnose and xylose, the main monomeric sugar constituents of pectin. An analysis of the corresponding transcriptomes revealed expression of 46 genes encoding pectinolytic enzymes. Their transcriptional profiles are discussed in detail and a cascade model of pectin degradation is proposed
    The growing VAO flavoprotein family
    Leferink, N.G.H. ; Heuts, D.P.H.M. ; Fraaije, M.W. ; Berkel, W.J.H. van - \ 2008
    Archives of Biochemistry and Biophysics 474 (2008)2. - ISSN 0003-9861 - p. 292 - 301.
    gamma-lactone dehydrogenase - vanillyl-alcohol oxidase - berberine bridge enzyme - para-cresol methylhydroxylase - covalently-bound flavin - vitamin-c biosynthesis - hydroxylase genes ehya/ehyb - site-directed mutagenesis - 6-hydroxy-d-nicotine oxidase - ascorbic-
    The VAO flavoprotein family is a rapidly growing family of oxidoreductases that favor the covalent binding of the FAD cofactor. In this review we report on the catalytic properties of some newly discovered VAO family members and their mode of flavin binding. Covalent binding of the flavin is a self-catalytic post-translational modification primarily taking place in oxidases. Covalent flavinylation increases the redox potential of the cofactor and thus its oxidation power. Recent findings have revealed that some members of the VAO family anchor the flavin via a dual covalent linkage (6-S-cysteinyl-8alpha-N1-histidyl FAD). Some VAO-type aldonolactone oxidoreductases favor the non-covalent binding of the flavin cofactor. These enzymes act as dehydrogenases, using cytochrome c as electron acceptor.
    Production and characterization of a thermostable L-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus
    Machielsen, M.P. ; Oost, J. van der - \ 2006
    FEBS Journal 273 (2006)12. - ISSN 1742-464X - p. 2722 - 2729.
    containing alcohol-dehydrogenase - site-directed mutagenesis - escherichia-coli - horikoshii - proteins - binding - identification - histidine-90 - degradation - catabolism
    The gene encoding a threonine dehydrogenase (TDH) has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The Pf-TDH protein has been functionally produced in Escherichia coli and purified to homogeneity. The enzyme has a tetrameric conformation with a molecular mass of ¿ 155 kDa. The catalytic activity of the enzyme increases up to 100°C, and a half-life of 11 min at this temperature indicates its thermostability. The enzyme is specific for NAD(H), and maximal specific activities were detected with l-threonine (10.3 U·mg-1) and acetoin (3.9 U·mg-1) in the oxidative and reductive reactions, respectively. Pf-TDH also utilizes l-serine and d-threonine as substrate, but could not oxidize other l-amino acids. The enzyme requires bivalent cations such as Zn2+ and Co 2+ for activity and contains at least one zinc atom per subunit. Km values for l-threonine and NAD+ at 70°C were 1.5 mm and 0.055 mm, respectively
    A new group of exo-acting family 28 glycoside hydrolases of Aspergillus niger that are involved in pectin degradation
    Martens-Uzunova, E.S. ; Zandleven, J.S. ; Benen, J.A.E. ; Awad, H. ; Kools, H.J. ; Beldman, G. ; Voragen, A.G.J. ; Berg, J.A. van den; Schaap, P.J. - \ 2006
    Biochemical Journal 400 (2006). - ISSN 0264-6021 - p. 43 - 52.
    site-directed mutagenesis - endopolygalacturonase-ii - xylogalacturonan hydrolase - rhamnogalacturonan regions - sequence alignment - crystal-structure - aculeatus - enzyme - gene - exopolygalacturonase
    The fungus Aspergillus niger is an industrial producer of pectin degrading enzymes. The recent solving of the genomic sequence of A. niger allowed an inventory of the entire genome of the fungus for potential carbohydrate degrading enzymes. By applying bioinformatics tools 12 new genes putatively encoding family 28 glycoside hydrolases were identified. Seven of the newly discovered genes form a new gene group, which we show to encode exo-acting pectinolytic glycoside hydrolases. This group includes four exo-polygalacturonan hydrolases (PGAX, PGXA, PGXB and PGXC) and three putative exo-rhamnogalacturonan hydrolases (RGXA, RGXB and RGXC). Biochemical identification using polygalacturonic acid and xylogalacturonan as substrates demonstrated that indeed PGXB and PGXC act as exopolygalacturonases while PGXA acts as an exo-xylogalacturonan hydrolase. The expression levels of all 21 genes were assessed by microarray analysis. The results from this study demonstrate that exo-acting glycoside hydrolases play a prominent role in pectin degradation.
    Necrotizing activity of five Botrytis cinerea endopolygalacturonases produced in Pichia pastoris
    Kars, I. ; Krooshof, G.H. ; Wagemakers, L. ; Joosten, R. ; Benen, J.A.E. ; Kan, J.A.L. van - \ 2005
    The Plant Journal 43 (2005)2. - ISSN 0960-7412 - p. 213 - 225.
    polygalacturonase-inhibiting proteins - site-directed mutagenesis - aspergillus-niger - cell-wall - sclerotinia-sclerotiorum - gene-expression - plant-pathogens - cutinase-a - fungal - tomato
    Five Botrytis cinerea endopolygalacturonase enzymes (BcPGs) were individually expressed in Pichia pastoris, purified to homogeneity and biochemically characterized. While the pH optima of the five enzymes were similar (approximately pH 4.5) the maximum activity of individual enzymes differed significantly. For hydrolysis of polygalacturonic acid (PGA), the Vmax,app ranged from 10 to 900 U mg1, while the Km,app ranged from 0.16 to 0.6 mg ml1. Although all BcPGs are true endopolygalacturonases, they apparently have different modes of action. PGA hydrolysis by BcPG1, BcPG2 and BcPG4 leads to the transient accumulation of oligomers with DP <7, whereas PGA hydrolysis by BcPG3 and BcPG6 leads to the immediate accumulation of monomers and dimers. The necrotizing activity (NA) of all BcPGs was tested separately in tomato, broad bean and Arabidopsis thaliana. They showed different NAs on these plants. BcPG1 and BcPG2 possessed the strongest NA as tissue collapse was observed within 10 min after infiltration of broad bean leaves. The amino acid (aa) D192A substitution in the active site of BcPG2 not only abolished enzyme activity but also the NA, indicating that the NA is dependent on enzyme activity. Furthermore, deletion of the Bcpg2 gene in B. cinerea resulted in a strong reduction in virulence on tomato and broad bean. Primary lesion formation was delayed by approximately 24 h and the lesion expansion rate was reduced by 50¿85%. These data indicate that BcPG2 is an important virulence factor for B. cinerea
    Characterisation and mode of action of an exopolygalacturonase from the hyperthermophilic bacterium Thermotoga maritime
    Kluskens, L.D. ; Alebeek, G.J.W.M. van; Walther, J. ; Voragen, A.G.J. ; Vos, W.M. de; Oost, J. van der - \ 2005
    FEBS Journal 272 (2005)21. - ISSN 1742-464X - p. 5464 - 5473.
    site-directed mutagenesis - aspergillus-niger - crystal-structure - butyrivibrio-fibrisolvens - endopolygalacturonase ii - subsite affinities - genome sequence - bovine rumen - sp-nov - pectin
    An intracellular pectinolytic enzyme, PelB (TM0437), from the hyperthermophilic bacterium Thermotoga maritima was functionally produced in Escherichia coli and purified to homogeneity. PelB belongs to family 28 of the glycoside hydrolases, consisting of pectin-hydrolysing enzymes. As one of the few bacterial exopolygalacturonases, it is able to remove monogalacturonate units from the nonreducing end of polygalacturonate. Detailed characterization of the enzyme showed that PelB is highly thermo-active and thermostable, with a melting temperature of 105 °C and a temperature optimum of 80 °C, the highest described to date for hydrolytic pectinases. PelB showed increasing activity on oligosaccharides with an increasing degree of polymerization. The highest activity was found on the pentamer (1000 U·mg1). In addition, the affinity increased in conjunction with the length of the oligoGalpA chain. PelB displayed specificity for saturated oligoGalpA and was unable to degrade unsaturated or methyl-esterified oligoGalpA. Analogous to the exopolygalacturonase from Aspergillus tubingensis, it showed low activity with xylogalacturonan. Calculations on the subsite affinity revealed the presence of four subsites and a high affinity for GalpA at subsite +1, which is typical of exo-active enzymes. The physiological role of PelB and the previously characterized exopectate lyase PelA is discussed.
    The molecular origin of the thiamin diphosphate-induced spectral bands of ThDP-dependent enzymes
    Kovina, M.V. ; Kok, A. ; Sevostyanova, I.A. ; Khailova, L.S. ; Belkina, N.V. ; Kochetov, G.A. - \ 2004
    Proteins : Structure, Function, and Bioinformatics 56 (2004)2. - ISSN 0887-3585 - p. 338 - 345.
    pyruvate-dehydrogenase complex - site-directed mutagenesis - transketolase-catalyzed reaction - yeast transketolase - binding-site - active-centers - bakers-yeast - pyrophosphate - decarboxylase - substrate
    New and previously published data on a variety of ThDP-dependent enzymes such as baker's yeast transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase from pigeon breast muscle, bovine heart, bovine kidney, Neisseria meningitidis and E. coli show their spectral sensitivity to ThDP binding. Although ThDP-induced spectral changes are different for different enzymes, their universal origin is suggested as being caused by the intrinsic absorption of the pyrimidine ring of ThDP, bound in different tautomeric forms with different enzymes. Non-enzymatic models with pyrimidine-like compounds indicate that the specific protein environment of the aminopyrimidine ring of ThDP determines its tautomeric form and therefore the changeable features of the inducible effect. A polar environment causes the prevalence of the aminopyrimidine tautomeric form (short wavelength region is affected). For stabilization of the iminopyrimidine tautomeric form (both short- and long-wavelength regions are affected) two factors appear essential: (i) a nonpolar environment and (ii) a conservative carboxyl group of a specific glutamate residue interacting with the N1 atom of the aminopyrimidine ring. The two types of optical effect depend in a different way upon the pH, in full accordance with the hypothesis tested. From these studies it is concluded that the inducible optical rotation results from interaction of the aminopyrimidine ring with its asymmetric environment and is defined by the protonation state of N1 and the 4-nitrogen
    Enantiospecific (+)-and (-)germacrene D synthases, cloned from goldenrod, reveal a functionally active variant of the universal isoprenoid-biosynthesis aspartate-rich motif
    Prosser, I. ; Altug, I.G. ; Philips, A.L. ; König, W.A. ; Bouwmeester, H.J. ; Beale, M. - \ 2004
    Archives of Biochemistry and Biophysics 432 (2004)2. - ISSN 0003-9861 - p. 136 - 144.
    site-directed mutagenesis - gamma-humulene synthase - delta-selinene synthase - artemisia-annua l - bacterial expression - germacrene-d - trichodiene synthase - cdna isolation - sesquiterpene cyclase - solidago-canadensis
    The naturally occurring, volatile sesquiterpene hydrocarbon germacrene D has strong effects on insect behaviour and genes encoding enzymes that produce this compound are of interest in the study of plant–insect interactions and in a number of biotechnological approaches to pest control. Goldenrod, Solidago canadensis, is unusual in that it produces both enantiomers of germacrene D. Two new sesquiterpene synthase cDNAs, designated Sc11 and Sc19, have been isolated from goldenrod and functional expression in Escherichia coli identified Sc11 as (+)-germacrene D synthase and Sc19 as (-)-germacrene D synthase. Thus, the enantiomers of germacrene D are the products of separate, but closely related (85% amino-acid identity), enzymes. Unlike other sesquiterpene synthases and the related monoterpene synthases and prenyl transferases, which contain the characteristic amino-acid motif DDXX(D, E), Sc11 is unusual in that this motif occurs as 303NDTYD. Mutagenesis of this motif to 303DDTYD gave rise to an enzyme that fully retained (+)-germacrene D synthase activity. The converse mutation in Sc19 (D303N) resulted in a less efficient but functional enzyme. Mutagenesis of position 303 to glutamate in both enzymes resulted in loss of activity. These results indicate that the magnesium ion-binding role of the first aspartate in the DDXXD motif may not be as critical as previously thought. Further amino-acid sequence comparisons and molecular modelling of the enzyme structures revealed that very subtle changes to the active site of this family of enzymes are required to alter the reaction pathway to form, in this case, different enantiomers from the same enzyme-bound carbocationic intermediate.
    Covalent flavinylation enhances the oxidative power of vanillyl-alcohol oxidase
    Fraaije, M.W. ; Heuvel, R.H.H. van den; Mattevi, A. ; Berkel, W.J.H. van - \ 2003
    Journal of Molecular Catalysis. B, Enzymatic 21 (2003)1-2. - ISSN 1381-1177 - p. 43 - 46.
    p-cresol methylhydroxylase - site-directed mutagenesis - fad-binding - redox properties - mononucleotide - attachment
    Vanillyl-alcohol oxidase (VAO) from Penicillium simplicissimum is an inducible flavoprotein that is active with a wide range of phenolic compounds. The enzyme is the prototype of a newly recognized family of structurally related oxidoreductases, whose members share a conserved FAD-binding domain. The flavin cofactor in VAO is covalently linked to His422 of the cap domain. Studies from His422 variants revealed that deletion of the histidyl-flavin bond does not result in any significant structural change. However, the covalent interaction increases the redox potential of the flavin, facilitating substrate oxidation. His61, located in the FAD-binding domain, is involved in the autocatalytic process of covalent flavinylation. This could be nicely demonstrated by creating the H61T mutant enzyme which binds the flavin in a non-covalently mode. Similar to the noncovalent His422 variants, H61T is 10-fold less active than wild-type VAO. From this and the similar crystal structures of apo and holo H61T it is concluded that the FAD binds to a preorganized binding site where His61 activates His422 for autocatalytic flavinylation. (C) 2002 Published by Elsevier Science B.V.
    Identification and molecular characterization of a novel type of alpha-galactosidase from Pyrococcus furiosus
    Lieshout, J.F.T. van; Verhees, C.H. ; Ettema, T.J.G. ; Sar, S. van der; Imamura, H. ; Matsuzawa, H. ; Oost, J. van der; Vos, W.M. de - \ 2003
    Biocatalysis and Biotransformation 21 (2003)4-5. - ISSN 1024-2422 - p. 243 - 252.
    geobacillus-stearothermophilus t-6 - catalytically essential residues - site-directed mutagenesis - adolescentis dsm 20083 - biochemical-characterization - escherichia-coli - hyperthermophilic archaebacterium - thermococcus-litoralis - sulfolobus-solfataricus - l-
    An -galactosidase gene from Pyrococcus furiosus was identified, cloned and functionally expressed in Escherichia coli. It is the first -galactosidase from a hyperthermophilic archaeon described to date. The gene encodes a unique amino acid sequence compared to other -galactosidases. Highest homology was found with -amylases classified in family 57 of glycoside hydrolases. The 364 amino acid protein had a calculated mass of 41.6 kDa. The recombinant -galactosidase specifically catalyzed the hydrolysis of para-nitrophenyl--galactopyranoside, and to some extent that of melibiose and raffinose. The enzyme proved to be an extremely thermo-active and thermostable -galactosidase with a temperature optimum of 115°C and a half-life time of 15 hours at 100°C. The pH optimum is between 5.0 and 5.5. Sequence analysis showed four conserved carboxylic residues. Site-directed mutagenesis was applied to identify the potential catalytic residues. Glu117Ala showed decreased enzyme activity, which could be rescued by the addition of azide or formate. It is concluded that glutamate 117 is the catalytic nucleophile, whereas the acid/base catalyst remains to be identified.
    Structural insights into the processivity of endopolygalacturonase I from Aspergillus niger
    Pouderoyen, G. van; Snijder, H.J. ; Benen, J.A.E. ; Dijkstra, B.W. - \ 2003
    FEBS Letters 554 (2003). - ISSN 0014-5793 - p. 462 - 466.
    site-directed mutagenesis - acid-sequence similarities - crystal-structure - crystallographic evidence - angstrom resolution - protein - complex - polygalacturonase - classification - identification
    Endopolygalacturonase I is a processive enzyme, while the 60% sequence identical endopolygalacturonase II is not. The 1.70 Angstrom resolution crystal structure of endopolygalacturonase I reveals a narrowed substrate binding cleft. In addition, Arg96, a residue in this cleft previously shown to be critical for processivity, interacts with the substrate mimics glycerol and sulfate in several well-defined conformations in the six molecules in the asymmetric unit. From this we conclude that both Arg96 and the narrowed substrate binding cleft contribute to retaining the substrate while it moves through the active site after a cleavage event has occurred. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
    Identification of amino acid residues critical for catalysis and stability in Aspergillus niger family 1 pectin lyase A
    Sanchez-Torres, P. ; Visser, J. ; Benen, J.A.E. - \ 2003
    Biochemical Journal 370 (2003). - ISSN 0264-6021 - p. 331 - 337.
    site-directed mutagenesis - plant virulence factor - pectate lyase - endopolygalacturonase ii - 3-dimensional structure - gene family - enzymes - pathogenesis - expression - erwinia
    Site-directed-mutagenesis studies were performed on family 1 pectin lyase A (PL1A) from Aspergillus niger to gain insight into the reaction mechanism for the pectin lyase-catalysed beta-elimination cleavage of methylesterified polygalacturonic acid and to stabilize the enzyme at slightly basic pH. On the basis of the three-dimensional structures of PL1A [Mayans, Scott, Connerton, Gravesen, Benen, Visser, Pickersgill and Jenkins (1997) Structure 5, 677-689] and the modelled enzyme-substrate complex of PL1B [Herron, Benen, Scavetta, Visser and Jurnak (2000) Proc. Nail. Acad. Sci. U.S.A. 97, 8762-8769], Asp(154), Arg(176), Arg(236) and Lys(239) were mutagenized. Substituting Arg(236) with alanine or lysine rendered the enzyme completely inactive, and mutagenesis of Arg(176) and Lys(239) severely affected catalysis. The Asp(154) --> Arg and Asp(154) --> Glu mutant enzymes were only moderately impaired in respect of catalysis. The results strongly indicate that Arg(236), which is sandwiched between Arg(176) and Lys(239), would initiate the reaction upon enzyme-substrate interaction, through the abstraction of the proton at C-5 of the galacturonopyranose ring. The positively charged residues Arg(176) and Lys(239) are responsible for lowering the pK(a) of Arg(236). Arg(176) and Lys(239) are maintained in a charged state by interacting with Asp(154) or bulk solvent respectively. The deprotonation of the Asp(186)-Asp(221) pair was proposed to be responsible for a pH-driven conformational change of PL1A [Mayans, Scott, Connerton, Gravesen, Benen, Visser, Pickersgill and Jenkins (1997) Structure 5, 677-689]. Substitution of Asp(186) and Asp(221) by Asn(186) and Asti(221) was expected to stabilize the enzyme. However, the Asp(186) --> Asn/Asp(221) --> Asn enzyme appeared less stable than the wild-type enzyme, even at pH 6.0, as evidenced by fluorescence studies. This demonstrates that the pH-dependent conformational change is not driven by deprotonation of the Asp(186) --> Asp(221) pair.
    Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specifity.
    Tamer, M.K. el; Lucker, J. ; Bosch, D. ; Verhoeven, H.A. ; Verstappen, F.W.A. ; Schwab, W. ; Tunen, A.J. van; Voragen, A.G.J. ; Maagd, R.A. de; Bouwmeester, H.J. - \ 2003
    Archives of Biochemistry and Biophysics 411 (2003). - ISSN 0003-9861 - p. 196 - 203.
    site-directed mutagenesis - 5-epi-aristolochene synthase - trichodiene synthase - linalool synthase - germacrene-a - biosynthesis - expression - biology
    Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a P-pinene synthase named Cl(-)betaPINS, a gamma-terpinene synthase named ClgammaTS, and two limonene synthases named Cl(+)LIMS1 and Cl(+)LIMS2 were recently isolated [J. Lucker et al., Eur. J. Biochem. 269 (2002) 3160]. The aim of our work in this study was to identify domains within these monoterpene synthase enzymes determining the product specificity. Domain swapping experiments between Cl(-)betaPINS and ClgammaTS and between Cl(+)LIMS2 and ClyTS were conducted. We found that within the C-terminal domain of these monoterpene synthases, a region comprising 200 amino acids, of which 41 are different between Cl(-)betaPINS and ClgammaTS, determines the specificity for the formation of P-pinene or gamma-terpinene, respectively, while another region localized further downstream is required for a chimeric enzyme to yield products in the same ratio as in the wild-type ClgammaTS. For Cl(+)LIMS2, the two domains together appear to be sufficient for its enzyme specificity, but many chimeras were inactive probably due to the low homology with ClyTS. Molecular modeling was used to further pinpoint the amino acids responsible for the differences in product specificity of ClyTS and Cl(-)betaPINS. (C) 2003 Elsevier Science (USA). All rights reserved.
    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.
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