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Ivonne M.c.m. Rietjens - One of the best experts on this subject based on the ideXlab platform.
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TEAC antioxidant activity of 4-Hydroxybenzoates.
Free radical biology & medicine, 1999Co-Authors: Bozena Tyrakowska, H Szymusiak, Ans E.m.f. Soffers, Marelle G. Boersma, Sjef Boeren, Karianto Leman, Jacques Vervoort, Ivonne M.c.m. RietjensAbstract:The influence of pH, intrinsic electron donating capacity, and intrinsic hydrogen atom donating capacity on the antioxidant potential of series of hydroxy and fluorine substituted 4-Hydroxybenzoates was investigated experimentally and also on the basis of computer calculations. The pH-dependent behavior of the compounds in the TEAC assay revealed different antioxidant behavior of the nondissociated monoanionic form and the deprotonated dianionic form of the 4-Hydroxybenzoates. Upon deprotonation the radical scavenging ability of the 4-Hydroxybenzoates increases significantly. For mechanistic comparison a series of fluorobenzoates was synthesized and included in the studies. The fluorine substituents were shown to affect the proton and electron donating abilities of 4-Hydroxybenzoate in the same way as hydroxyl substituents. In contrast, the fluorine substituents influenced the TEAC value and the hydrogen atom donating capacity of 4-Hydroxybenzoate in a way different from the hydroxyl moieties. Comparison of these experimental data to computer-calculated characteristics indicates that the antioxidant behavior of the monoanionic forms of the 4-Hydroxybenzoates is not determined by the tendency of the molecule to donate an electron, but by its ability to donate a hydrogen atom. Altogether, the results explain qualitatively and quantitatively how the number and position of OH moieties affect the antioxidant behavior of 4-Hydroxybenzoates.
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Catabolism of 4-Hydroxybenzoate in proceeds through initial oxidative decarboxylation by a FAD-dependent 4-Hydroxybenzoate 1-hydroxylase
Fems Microbiology Letters, 1994Co-Authors: W Vanberkel, Jacques Vervoort, M Eppink, Wouter J. Middelhoven, Ivonne M.c.m. RietjensAbstract:The first two steps in the catabolism of 4-Hydroxybenzoate by the ascomycetous yeast Candida parapsilosis CBS604 were investigated. In contrast to the well-known bacterial pathways and to what was previously assumed, metabolism of 4-Hydroxybenzoate in C. parapsilosis proceeds through initial oxidative decarboxylation to give 1,4-dihydroxybenzene. This reaction is catalyzed by a NAD(P)H and FAD-dependent 4-Hydroxybenzoate 1-hydroxylase. Further metabolism of 1,4-dihydroxybenzene to the ring-fission substrate 1,2,4-trihydroxybenzene is catalyzed by a NADPH-specific FAD-dependent aromatic hydroxylase acting on phenolic compounds. 19F-NMR experiments with cell extracts and 2-fluoro-4-Hydroxybenzoate as the model compound confirm this metabolic pathway and exclude the alternative pathway proceeding through initial 3-hydroxylation followed by oxidative decarboxylation in the second step.
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Catabolism of 4-Hydroxybenzoate in Candida parapsilosis proceeds through initial oxidative decarboxylation by a FAD-dependent 4-Hydroxybenzoate 1-hydroxylase
FEMS microbiology letters, 1994Co-Authors: Willem J. H. Van Berkel, M Eppink, Jacques Vervoort, Wouter J. Middelhoven, Ivonne M.c.m. RietjensAbstract:The first two steps in the catabolism of 4-Hydroxybenzoate by the ascomycetous yeast Candida parapsilosis CBS604 were investigated. In contrast to the well-known bacterial pathways and to what was previously assumed, metabolism of 4-Hydroxybenzoate in C. parapsilosis proceeds through initial oxidative decarboxylation to give 1,4-dihydroxybenzene. This reaction is catalyzed by a NAD(P)H and FAD-dependent 4-Hydroxybenzoate 1-hydroxylase. Further metabolism of 1,4-dihydroxybenzene to the ring-fission substrate 1,2,4-trihydroxybenzene is catalyzed by a NADPH-specific FAD-dependent aromatic hydroxylase acting on phenolic compounds. 19F-NMR experiments with cell extracts and 2-fluoro-4-Hydroxybenzoate as the model compound confirm this metabolic pathway and exclude the alternative pathway proceeding through initial 3-hydroxylation followed by oxidative decarboxylation in the second step.
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Frontier orbital study on the 4-Hydroxybenzoate-3-hydroxylase-dependent activity with benzoate derivatives.
European journal of biochemistry, 1992Co-Authors: Jacques Vervoort, Ivonne M.c.m. Rietjens, W J H Van Berkel, Cees VeegerAbstract:Based on molecular orbital computer calculations the present paper provides a new hypothesis for catalytic characteristics of 4-Hydroxybenzoate-3-hydroxylase (EC 1.14.13.2). A clear correlation between In k,,, for the conversion of a series of 4-hydroxylated substrates and their E(HOM0) leads to the hypothesis that Frontier orbital HOMO characteristics [E(HOMO) and HOMO density on C3] of the substrates are the predominant factor in regulating the fate of a benzoate derivative at the active site of the enzyme. The HOMO characteristics can be used to explain whether a compound will be converted by the enzyme or merely acts as an effector. Furthermore the hypothesis provides quantitative theoretical support for a catalytic mechanism in which the substrate reacts in its dianionic form and for a mechanism in which the electrophilic attack of the C(4a)-peroxyflavin, or of the hydroxyl radical derived from it, on the benzoate dianion is the rate limiting step in catalysis at pH 8, 25 "C. Finally, it is demonstrated that the hypothesis can be used as a basis for the formulation of working hypotheses in future research, investigating the conversion and regioselective orientation of the various possible substrates in the active site of the wild-type 4-Hydroxybenzoate-3-hydroxylase, its mutants as well as of various other flavin-dependent aromatic hydroxylases, such as for example 3hydroxybenzoate-4-hydroxylase (EC 1.14.13.23), 3-hydroxybenzoate-6-hydroxylase (EC 1.14.13.24) and phenol hydroxylase (EC 1.14.13.7). The flavoprotein 4-Hydroxybenzoate-3-hydroxylase is
Jacques Vervoort - One of the best experts on this subject based on the ideXlab platform.
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TEAC antioxidant activity of 4-Hydroxybenzoates.
Free radical biology & medicine, 1999Co-Authors: Bozena Tyrakowska, H Szymusiak, Ans E.m.f. Soffers, Marelle G. Boersma, Sjef Boeren, Karianto Leman, Jacques Vervoort, Ivonne M.c.m. RietjensAbstract:The influence of pH, intrinsic electron donating capacity, and intrinsic hydrogen atom donating capacity on the antioxidant potential of series of hydroxy and fluorine substituted 4-Hydroxybenzoates was investigated experimentally and also on the basis of computer calculations. The pH-dependent behavior of the compounds in the TEAC assay revealed different antioxidant behavior of the nondissociated monoanionic form and the deprotonated dianionic form of the 4-Hydroxybenzoates. Upon deprotonation the radical scavenging ability of the 4-Hydroxybenzoates increases significantly. For mechanistic comparison a series of fluorobenzoates was synthesized and included in the studies. The fluorine substituents were shown to affect the proton and electron donating abilities of 4-Hydroxybenzoate in the same way as hydroxyl substituents. In contrast, the fluorine substituents influenced the TEAC value and the hydrogen atom donating capacity of 4-Hydroxybenzoate in a way different from the hydroxyl moieties. Comparison of these experimental data to computer-calculated characteristics indicates that the antioxidant behavior of the monoanionic forms of the 4-Hydroxybenzoates is not determined by the tendency of the molecule to donate an electron, but by its ability to donate a hydrogen atom. Altogether, the results explain qualitatively and quantitatively how the number and position of OH moieties affect the antioxidant behavior of 4-Hydroxybenzoates.
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purification and properties of 4 hydroxybenzoate 1 hydroxylase decarboxylating a novel flavin adenine dinucleotide dependent monooxygenase from candida parapsilosis cbs604
Journal of Bacteriology, 1997Co-Authors: Michel H. M. Eppink, Sjef Boeren, Jacques Vervoort, W J H Van BerkelAbstract:A novel flavoprotein monooxygenase, 4-Hydroxybenzoate 1-hydroxylase (decarboxylating), from Candida parapsilosis CBS604 was purified to apparent homogeneity. The enzyme is induced when the yeast is grown on either 4-Hydroxybenzoate, 2,4-dihydroxybenzoate, or 3,4-dihydroxybenzoate as the sole carbon source. The purified monooxygenase is a monomer of about 50 kDa containing flavin adenine dinucleotide as weakly bound cofactor. 4-Hydroxybenzoate 1-hydroxylase from C. parapsilosis catalyzes the oxidative decarboxylation of a wide range of 4-Hydroxybenzoate derivatives with the stoichiometric consumption of NAD(P)H and oxygen. Optimal catalysis is reached at pH 8, with NADH being the preferred electron donor. By using (18)O2, it was confirmed that the oxygen atom inserted into the product 1,4-dihydroxybenzene is derived from molecular oxygen. 19F nuclear magnetic resonance spectroscopy revealed that the enzyme catalyzes the conversion of fluorinated 4-Hydroxybenzoates to the corresponding hydroquinones. The activity of the enzyme is strongly inhibited by 3,5-dichloro-4-Hydroxybenzoate, 4-hydroxy-3,5-dinitrobenzoate, and 4-hydroxyisophthalate, which are competitors with the aromatic substrate. The same type of inhibition is exhibited by chloride ions. Molecular orbital calculations show that upon deprotonation of the 4-hydroxy group, nucleophilic reactivity is located in all substrates at the C-1 position. This, and the fact that the enzyme is highly active with tetrafluoro-4-Hydroxybenzoate and 4-hydroxy-3-nitrobenzoate, suggests that the phenolate forms of the substrates play an important role in catalysis. Based on the substrate specificity, a mechanism is proposed for the flavin-mediated oxidative decarboxylation of 4-Hydroxybenzoate.
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Flavin Motion in p-Hydroxybenzoate Hydroxylase. Substrate and Effector Specificity of the Tyr222Ala Mutant
FEBS Journal, 1996Co-Authors: Frank J. T. Van Der Bolt, Jacques Vervoort, Willem J. H. Van BerkelAbstract:The side chain of Tyr222 in p -hydroxybenzoate hydroxylase interacts with the carboxy moiety of the substrate. Studies on the Tyr222Phe mutant, [F222]p -hydroxybenzoate hydroxylase, have shown that disruption of this interaction hampers the hydroxylation of 4-Hydroxybenzoate. Tyr222 is possibly involved in flavin motion, which may facilitate the exchange of substrate and product during catalysis. To elucidate the function of Tyr222 in more detail, in the present study the substrate and effector specificity of the Tyr222Ala mutant, [A222]p -hydroxybenzoate hydroxylase, was investigated. Replacement of Tyr222 by Ala impairs the binding of the physiological substrate 4-Hydroxybenzoate and the substrate analog 4-aminobenzoate. With these compounds, [A222]p -hydroxybenzoate hydroxylase mainly acts as a NADPH oxidase. [A222]p -hydroxybenzoate hydroxylase tightly interacts with 2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate. Crystallographic data [Schreuder, H. A., Mattevi, A., Oblomova, G., Kalk, K. H., Hoi, W. G. J., van der Bolt, F. J. T. & van Berkel, W. J. H (1994) Biochemistry 33, 10161–10170] suggest that this is due to motion of the flavin ring out of the active site, allowing hydrogen-bond interaction between the 2-hydroxy group of the substrate analogs and N3 of the flavin. [A222]p -Hydroxybenzoate hydroxylase produces about 0.6 mol 2,3,4-trihydroxybenzoate from 2,4-dihy-droxybenzoate/mol NADPH oxidized. This indicates that reduction of the Tyr222Ala mutant shifts the equilibrium of flavin conformers towards the productive ‘in’ position. [A222]p -Hydroxybenzoate hydroxylase converts 2-fluoro-4-Hydroxybenzoate to 2-fluoro-3,4-dihydroxybenzoate. The regioselectivity of hydroxylation suggests that [A222]p -hydroxybenzoate hydroxylase binds the fluorinated substrate in the same orientation as wild-type. Spectral studies suggest that wild-type and [A222]p -hydroxybenzoate hydroxylase bind 2-fluoro-4-Hydroxybenzoate in the phenolate form with the flavin ring preferring the ‘out’ conformation. Despite activation of the fluorinated substrate and in contrast to the wild-type enzyme, [A222]p -hydroxybenzoate hydroxylase largely produces hydrogen peroxide. The effector specificity of p -hydroxybenzoate hydroxylase is not changed by the Tyr222-Ala replacement. This supports the idea that the effector specificity is mainly dictated by the protein-substrate interactions at the re -side of the flavin ring.
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Catabolism of 4-Hydroxybenzoate in proceeds through initial oxidative decarboxylation by a FAD-dependent 4-Hydroxybenzoate 1-hydroxylase
Fems Microbiology Letters, 1994Co-Authors: W Vanberkel, Jacques Vervoort, M Eppink, Wouter J. Middelhoven, Ivonne M.c.m. RietjensAbstract:The first two steps in the catabolism of 4-Hydroxybenzoate by the ascomycetous yeast Candida parapsilosis CBS604 were investigated. In contrast to the well-known bacterial pathways and to what was previously assumed, metabolism of 4-Hydroxybenzoate in C. parapsilosis proceeds through initial oxidative decarboxylation to give 1,4-dihydroxybenzene. This reaction is catalyzed by a NAD(P)H and FAD-dependent 4-Hydroxybenzoate 1-hydroxylase. Further metabolism of 1,4-dihydroxybenzene to the ring-fission substrate 1,2,4-trihydroxybenzene is catalyzed by a NADPH-specific FAD-dependent aromatic hydroxylase acting on phenolic compounds. 19F-NMR experiments with cell extracts and 2-fluoro-4-Hydroxybenzoate as the model compound confirm this metabolic pathway and exclude the alternative pathway proceeding through initial 3-hydroxylation followed by oxidative decarboxylation in the second step.
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Catabolism of 4-Hydroxybenzoate in Candida parapsilosis proceeds through initial oxidative decarboxylation by a FAD-dependent 4-Hydroxybenzoate 1-hydroxylase
FEMS microbiology letters, 1994Co-Authors: Willem J. H. Van Berkel, M Eppink, Jacques Vervoort, Wouter J. Middelhoven, Ivonne M.c.m. RietjensAbstract:The first two steps in the catabolism of 4-Hydroxybenzoate by the ascomycetous yeast Candida parapsilosis CBS604 were investigated. In contrast to the well-known bacterial pathways and to what was previously assumed, metabolism of 4-Hydroxybenzoate in C. parapsilosis proceeds through initial oxidative decarboxylation to give 1,4-dihydroxybenzene. This reaction is catalyzed by a NAD(P)H and FAD-dependent 4-Hydroxybenzoate 1-hydroxylase. Further metabolism of 1,4-dihydroxybenzene to the ring-fission substrate 1,2,4-trihydroxybenzene is catalyzed by a NADPH-specific FAD-dependent aromatic hydroxylase acting on phenolic compounds. 19F-NMR experiments with cell extracts and 2-fluoro-4-Hydroxybenzoate as the model compound confirm this metabolic pathway and exclude the alternative pathway proceeding through initial 3-hydroxylation followed by oxidative decarboxylation in the second step.
Caroline S Harwood - One of the best experts on this subject based on the ideXlab platform.
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4-hydroxybenzoyl coenzyme A reductase (dehydroxylating) is required for anaerobic degradation of 4-Hydroxybenzoate by Rhodopseudomonas palustris and shares features with molybdenum-containing hydroxylases.
Journal of bacteriology, 1997Co-Authors: Jane Gibson, Marilyn Dispensa, Caroline S HarwoodAbstract:The anaerobic degradation of 4-Hydroxybenzoate is initiated by the formation of 4-hydroxybenzoyl coenzyme A, with the next step proposed to be a dehydroxylation to benzoyl coenzyme A, the starting compound for a central pathway of aromatic compound ring reduction and cleavage. Three open reading frames, divergently transcribed from the 4-Hydroxybenzoate coenzyme A ligase gene, hbaA, were identified and sequenced from the phototrophic bacterium Rhodopseudomonas palustris. These genes, named hbaBCD, specify polypeptides of 17.5, 82.6, and 34.5 kDa, respectively. The deduced amino acid sequences show considerable similarities to a group of hydroxylating enzymes involved in CO, xanthine, and nicotine metabolism that have conserved binding sites for [2Fe-2S] clusters and a molybdenum cofactor. Cassette disruption of the hbaB gene yielded a mutant that was unable to grow anaerobically on 4-Hydroxybenzoate but grew normally on benzoate. The hbaB mutant cells did not accumulate [14C]benzoyl coenzyme A during short-term uptake of [14C]4-Hydroxybenzoate, but benzoyl coenzyme A was the major radioactive metabolite formed by the wild type. In addition, crude extracts of the mutant failed to convert 4-hydroxybenzoyl coenzyme A to benzoyl coenzyme A. This evidence indicates that the hbaBCD genes encode the subunits of a 4-hydroxybenzoyl coenzyme A reductase (dehydroxylating). The sizes of the specified polypeptides are similar to those reported for 4-hydroxybenzoyl coenzyme A reductase isolated from the denitrifying bacterium Thauera aromatica. The amino acid consensus sequence for a molybdenum cofactor binding site is in HbaC. This cofactor appears to be an essential component because anaerobic growth of R. palustris on 4-Hydroxybenzoate, but not on benzoate, was retarded unless 0.1 microM molybdate was added to the medium. Neither tungstate nor vanadate replaced molybdate, and tungstate competitively inhibited growth stimulation by molybdate.
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Repression of 4-Hydroxybenzoate transport and degradation by benzoate: a new layer of regulatory control in the Pseudomonas putida beta-ketoadipate pathway.
Journal of bacteriology, 1995Co-Authors: Nancy N Nichols, Caroline S HarwoodAbstract:Pseudomonas putida PRS2000 degrades the aromatic acids benzoate and 4-Hydroxybenzoate via two parallel sequences of reactions that converge at beta-ketoadipate, a derivative of which is cleaved to form tricarboxylic acid cycle intermediates. Structural genes (pca genes) required for the complete degradation of 4-Hydroxybenzoate via the protocatechuate branch of the beta-ketoadipate pathway have been characterized, and a specific transport system for 4-Hydroxybenzoate has recently been described. To better understand how P. putida coordinates the processes of 4-Hydroxybenzoate transport and metabolism to achieve complete degradation, the regulation of pcaK, the 4-Hydroxybenzoate transport gene, and that of pcaF, a gene required for both benzoate and 4-Hydroxybenzoate degradation, were compared. Primer extension analysis and lacZ fusions showed that pcaK and pcaF, which are adjacent on the chromosome, are transcribed independently. PcaR, a transcriptional activator of several genes of the beta-ketoadipate pathway, is required for expression of both pcaF and pcaK, and the pathway intermediate beta-ketoadipate induces both genes. In addition to these expected regulatory elements, expression of pcaK, but not pcaF, is repressed by benzoate. This previously unrecognized layer of regulatory control in the beta-ketoadipate pathway appears to extend to the first two steps of 4-Hydroxybenzoate degradation, since levels of 4-Hydroxybenzoate hydroxylase and protocatechuate 3,4-dioxygenase activities were also depressed when cells were grown on a mixture of 4-Hydroxybenzoate and benzoate. The apparent consequence of benzoate repression is that cells degrade benzoate in preference to 4-Hydroxybenzoate. These findings indicate that 4-Hydroxybenzoate transport is an integral feature of the beta-ketoadipate pathway in P. putida and that transport plays a role in establishing the preferential degradation of benzoate over 4-Hydroxybenzoate. These results also demonstrate that there is communication between the two branches of the beta-ketoadipate pathway.
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identification of the pcarkf gene cluster from pseudomonas putida involvement in chemotaxis biodegradation and transport of 4 hydroxybenzoate
Journal of Bacteriology, 1994Co-Authors: Caroline S Harwood, Nancy N Nichols, Jayna L Ditty, Rebecca E ParalesAbstract:Pseudomonas putida PRS2000 is chemotactic to 4-Hydroxybenzoate and other aromatic acids. This behavioral response is induced when cells are grown on 4-Hydroxybenzoate or benzoate, compounds that are degraded via the beta-ketoadipate pathway. Isolation of a transposon mutant defective in 4-Hydroxybenzoate chemotaxis allowed identification of a new gene cluster designated pcaRKF. DNA sequencing, mutational analysis, and complementation studies revealed that pcaR encodes a regulatory protein required for induction of at least four of the enzymes of the beta-ketoadipate pathway and that pcaF encodes beta-ketoadipyl-coenzyme A thiolase, the last enzyme in the pathway. The third gene, pcaK, encodes a transporter for 4-Hydroxybenzoate, and this protein is also required for chemotaxis to aromatic acids. The predicted PcaK protein is 47 kDa in size, with a deduced amino acid sequence indicative of membership in the major facilitator superfamily of transport proteins. The protein, expressed in Escherichia coli, catalyzed 4-Hydroxybenzoate transport. In addition, whole cells of P. putida pcaK mutants accumulated 4-Hydroxybenzoate at reduced rates compared with that in wild-type cells. The pcaK mutation did not impair growth at the expense of 4-Hydroxybenzoate under most conditions; however, mutant cells grew somewhat more slowly than the wild type on 4-Hydroxybenzoate at a high pH. The finding that 4-Hydroxybenzoate chemotaxis can be disrupted without an accompanying effect on metabolism indicates that this chemotactic response is receptor mediated. It remains to be determined, however, whether PcaK itself is a chemoreceptor for 4-Hydroxybenzoate or whether it plays an indirect role in chemotaxis. These findings indicate that aromatic acid detection and transport are integral features of aromatic degradation pathways. Images
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4-Hydroxybenzoate-coenzyme A ligase from Rhodopseudomonas palustris: purification, gene sequence, and role in anaerobic degradation.
Journal of bacteriology, 1994Co-Authors: Jane Gibson, Marilyn Dispensa, G C Fogg, D T Evans, Caroline S HarwoodAbstract:Anaerobic metabolism of most aromatic acids is initiated by coenzyme A thioester formation. Rhodopseudomonas palustris grows well under anaerobic, phototrophic conditions with many aromatic acids, including benzoate and 4-Hydroxybenzoate, as a carbon source. A coenzyme A ligase that reacts with 4-Hydroxybenzoate was purified from 4-Hydroxybenzoate-grown cells of R. palustris. This enzyme required MgATP, reduced coenzyme A, and 4-Hydroxybenzoate, benzoate, or cyclohex-1,4-dienecarboxylate for optimal activity but also used phosphopantetheine, cyclohex-2,5-dienecarboxylate, and 4-fluorobenzoate at lower rates. The 4-Hydroxybenzoate-coenzyme A ligase differed in molecular characteristics from a previously described benzoate-coenzyme A ligase from R. palustris, and the two ligases did not cross-react immunologically. The gene encoding the 4-Hydroxybenzoate enzyme was cloned and sequenced. The deduced gene product showed about 20% amino acid identity with bacterial coenzyme A ligases involved in aerobic degradation of aromatic acids. An R. palustris mutant carrying a disrupted 4-Hydroxybenzoate-coenzyme A ligase gene was unable to grow with 4-Hydroxybenzoate under anaerobic conditions, indicating that the enzyme is essential for anaerobic degradation of this compound.
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Identification of the pcaRKF Gene Cluster from Pseudomonas Putida: Involvement in Chemotaxis, Biodegradation, and Transport of 4-Hydroxybenzoate
Journal of bacteriology, 1994Co-Authors: Caroline S Harwood, Nancy N Nichols, Jayna L Ditty, Min-kyung Kim, Rebecca E ParalesAbstract:Abstract Pseudomonas putida PRS2000 is chemotactic to 4-Hydroxybenzoate and other aromatic acids. This behavioral response is induced when cells are grown on 4-Hydroxybenzoate or benzoate, compounds that are degraded via the beta-ketoadipate pathway. Isolation of a transposon mutant defective in 4-Hydroxybenzoate chemotaxis allowed identification of a new gene cluster designated pcaRKF. DNA sequencing, mutational analysis, and complementation studies revealed that pcaR encodes a regulatory protein required for induction of at least four of the enzymes of the beta-ketoadipate pathway and that pcaF encodes beta-ketoadipyl-coenzyme A thiolase, the last enzyme in the pathway. The third gene, pcaK, encodes a transporter for 4-Hydroxybenzoate, and this protein is also required for chemotaxis to aromatic acids. The predicted PcaK protein is 47 kDa in size, with a deduced amino acid sequence indicative of membership in the major facilitator superfamily of transport proteins. The protein, expressed in Escherichia coli, catalyzed 4-Hydroxybenzoate transport. In addition, whole cells of P. putida pcaK mutants accumulated 4-Hydroxybenzoate at reduced rates compared with that in wild-type cells. The pcaK mutation did not impair growth at the expense of 4-Hydroxybenzoate under most conditions; however, mutant cells grew somewhat more slowly than the wild type on 4-Hydroxybenzoate at a high pH. The finding that 4-Hydroxybenzoate chemotaxis can be disrupted without an accompanying effect on metabolism indicates that this chemotactic response is receptor mediated. It remains to be determined, however, whether PcaK itself is a chemoreceptor for 4-Hydroxybenzoate or whether it plays an indirect role in chemotaxis. These findings indicate that aromatic acid detection and transport are integral features of aromatic degradation pathways.
Jane Montgomery - One of the best experts on this subject based on the ideXlab platform.
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evaluation of sodium 4 hydroxybenzoate as an hydroxyl radical trap using gas chromatography mass spectrometry and high performance liquid chromatography with electrochemical detection
Analytical Biochemistry, 1996Co-Authors: Line Stemarie, Daniel Boismenu, Luc Vachon, Jane MontgomeryAbstract:Abstract Molecular and tissue damage induced by reactive oxygen species is a serious consequence of the production of free radicals in biological systems. Biological markers produced by reactions with hydroxyl radicals are useful indices of free radical processesin vivo.In this respect, hydroxylation of aromatic compounds such as salicylate (2-hydroxybenzoate) has been used extensively as a measure of hydroxyl radical formation. 4-Hydroxybenzoate will also trap hydroxyl radicals with fewer of the complications for which salicylate has been criticized. We describe two sensitive and specific methods using gas chromatography–ion trap mass spectrometry and high-performance liquid chromatography with electrochemical detection for a number of these aromatic marker compounds in biological fluids. The use of an ion trap mass spectrometer provides enhanced sensitivity along with full mass spectral identification of the compounds of interest. 4-Hydroxybenzoate and salicylate were compared as hydroxyl radical traps (i) by determining relative hydroxyl radical trapping efficienciesin vitro,(ii) by measuring individual dihydroxybenzoate isomers in rat serum following intraperitoneal injection of either 2- or 4-Hydroxybenzoate, and (iii) by comparingin vivohydroxyl radical trapping using intrastriatal microdialysis in the rat. The techniques described have broad applications in the area of free radical biomedical research.
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Evaluation of Sodium 4-Hydroxybenzoate as an Hydroxyl Radical Trap Using Gas Chromatography–Mass Spectrometry and High-Performance Liquid Chromatography with Electrochemical Detection
Analytical biochemistry, 1996Co-Authors: Line Ste-marie, Daniel Boismenu, Luc Vachon, Jane MontgomeryAbstract:Abstract Molecular and tissue damage induced by reactive oxygen species is a serious consequence of the production of free radicals in biological systems. Biological markers produced by reactions with hydroxyl radicals are useful indices of free radical processes in vivo. In this respect, hydroxylation of aromatic compounds such as salicylate (2-hydroxybenzoate) has been used extensively as a measure of hydroxyl radical formation. 4-Hydroxybenzoate will also trap hydroxyl radicals with fewer of the complications for which salicylate has been criticized. We describe two sensitive and specific methods using gas chromatography–ion trap mass spectrometry and high-performance liquid chromatography with electrochemical detection for a number of these aromatic marker compounds in biological fluids. The use of an ion trap mass spectrometer provides enhanced sensitivity along with full mass spectral identification of the compounds of interest. 4-Hydroxybenzoate and salicylate were compared as hydroxyl radical traps (i) by determining relative hydroxyl radical trapping efficiencies in vitro, (ii) by measuring individual dihydroxybenzoate isomers in rat serum following intraperitoneal injection of either 2- or 4-Hydroxybenzoate, and (iii) by comparing in vivo hydroxyl radical trapping using intrastriatal microdialysis in the rat. The techniques described have broad applications in the area of free radical biomedical research.
W J H Van Berkel - One of the best experts on this subject based on the ideXlab platform.
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purification and properties of 4 hydroxybenzoate 1 hydroxylase decarboxylating a novel flavin adenine dinucleotide dependent monooxygenase from candida parapsilosis cbs604
Journal of Bacteriology, 1997Co-Authors: Michel H. M. Eppink, Sjef Boeren, Jacques Vervoort, W J H Van BerkelAbstract:A novel flavoprotein monooxygenase, 4-Hydroxybenzoate 1-hydroxylase (decarboxylating), from Candida parapsilosis CBS604 was purified to apparent homogeneity. The enzyme is induced when the yeast is grown on either 4-Hydroxybenzoate, 2,4-dihydroxybenzoate, or 3,4-dihydroxybenzoate as the sole carbon source. The purified monooxygenase is a monomer of about 50 kDa containing flavin adenine dinucleotide as weakly bound cofactor. 4-Hydroxybenzoate 1-hydroxylase from C. parapsilosis catalyzes the oxidative decarboxylation of a wide range of 4-Hydroxybenzoate derivatives with the stoichiometric consumption of NAD(P)H and oxygen. Optimal catalysis is reached at pH 8, with NADH being the preferred electron donor. By using (18)O2, it was confirmed that the oxygen atom inserted into the product 1,4-dihydroxybenzene is derived from molecular oxygen. 19F nuclear magnetic resonance spectroscopy revealed that the enzyme catalyzes the conversion of fluorinated 4-Hydroxybenzoates to the corresponding hydroquinones. The activity of the enzyme is strongly inhibited by 3,5-dichloro-4-Hydroxybenzoate, 4-hydroxy-3,5-dinitrobenzoate, and 4-hydroxyisophthalate, which are competitors with the aromatic substrate. The same type of inhibition is exhibited by chloride ions. Molecular orbital calculations show that upon deprotonation of the 4-hydroxy group, nucleophilic reactivity is located in all substrates at the C-1 position. This, and the fact that the enzyme is highly active with tetrafluoro-4-Hydroxybenzoate and 4-hydroxy-3-nitrobenzoate, suggests that the phenolate forms of the substrates play an important role in catalysis. Based on the substrate specificity, a mechanism is proposed for the flavin-mediated oxidative decarboxylation of 4-Hydroxybenzoate.
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4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3. Purification, characterization, gene cloning, sequence analysis and assignment of structural features determining the coenzyme specificity.
European journal of biochemistry, 1996Co-Authors: B. Seibold, Michel H. M. Eppink, W J H Van Berkel, M. Matthes, Franz Lingens, Rudolf MüllerAbstract:4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 was purified by five consecutive steps to apparent homogeneity. The enrichment was 50-fold with a yield of about 20%. The enzyme is a homodimeric flavoprotein monooxygenase with each 44-kDa polypeptide chain containing one FAD molecule as a rather weakly bound prosthetic group. In contrast to other 4-Hydroxybenzoate hydroxylases of known primary structure, the enzyme preferred NADH over NADPH as electron donor. The pH optimum for catalysis was pH 8.0 with a maximum turnover rate around 45 degrees C. Chloride ions were inhibitory, and competitive with respect to NADH. 4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 has a narrow substrate specificity. In addition to the transformation of 4-Hydroxybenzoate to 3,4-dihydroxybenzoate, the enzyme converted 2-fluoro-4-Hydroxybenzoate, 2-chloro-4-Hydroxybenzoate, and 2,4-dihydroxybenzoate. With all aromatic substrates, no uncoupling of hydroxylation was observed. The gene encoding 4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 was cloned in Escherichia coli. Nucleotide sequence analysis revealed an open reading frame of 1182 bp that corresponded to a protein of 394 amino acid residues. Upstream of the pobA gene, a sequence resembling an E. coli promoter was identified, which led to constitutive expression of the cloned gene in E. coli TG1. The deduced amino acid sequence of Pseudomonas sp. CBS3 4-Hydroxybenzoate hydroxylase revealed 53% identity with that of the pobA enzyme from Pseudomonas fluorescens for which a three-dimensional structure is known. The active-site residues and the fingerprint sequences associated with FAD binding are strictly conserved. This and the conservation of secondary structures implies that the enzymes share a similar three-dimensional fold. Based on an isolated region of sequence divergence and site-directed mutagenesis data of 4-Hydroxybenzoate hydroxylase from P. fluorescens, it is proposed that helix H2 is involved in determining the coenzyme specificity.
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Structure and function of mutant Arg44Lys of 4-Hydroxybenzoate hydroxylase: implications for NADPH binding.
European journal of biochemistry, 1995Co-Authors: Michel H. M. Eppink, Herman A. Schreuder, W J H Van BerkelAbstract:Arg44, located at the si-face side of the flavin ring in 4–hydroxybenzoate hydroxylase, was changed to lysine by site-specific mutagenesis. Crystals of [R44K]4-Hydroxybenzoate hydroxylase complexed with 4-Hydroxybenzoate diffract to 0.22-nm resolution. The structure of [R44K]4-Hydroxybenzoate hydroxylase is identical to the wild-type enzyme except for local changes in the vicinity of the mutation. The peptide unit between Ile43 and Lys44 is flipped by about 180° in 50% of the molecules. The φ,ψ angles in both the native and flipped conformation are outside the allowed regions and indicate a strained conformation. [R44K]4-Hydroxybenzoate hydroxylase has a decreased affinity for the flavin prosthetic group. This is ascribed to the lost interactions between the side chain of Arg44 and the diphosphoribose moiety of the FAD. The replacement of Arg44 by Lys does not change the position of the flavin ring which occupies the same interior position as in wild type. [R44K]4-Hydroxybenzoate hydroxylase fully couples flavin reduction to substrate hydroxylation. Stopped-flow kinetics showed that the effector role of 4-Hydroxybenzoate is largely conserved in the mutant. Replacement of Arg44 by Lys however affects NADPH binding, resulting in a low yield of the charge-transfer species between reduced flavin and NADP+. It is inferred from these data that Arg44 is indispensable for optimal catalysis.
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Frontier orbital study on the 4-Hydroxybenzoate-3-hydroxylase-dependent activity with benzoate derivatives.
European journal of biochemistry, 1992Co-Authors: Jacques Vervoort, Ivonne M.c.m. Rietjens, W J H Van Berkel, Cees VeegerAbstract:Based on molecular orbital computer calculations the present paper provides a new hypothesis for catalytic characteristics of 4-Hydroxybenzoate-3-hydroxylase (EC 1.14.13.2). A clear correlation between In k,,, for the conversion of a series of 4-hydroxylated substrates and their E(HOM0) leads to the hypothesis that Frontier orbital HOMO characteristics [E(HOMO) and HOMO density on C3] of the substrates are the predominant factor in regulating the fate of a benzoate derivative at the active site of the enzyme. The HOMO characteristics can be used to explain whether a compound will be converted by the enzyme or merely acts as an effector. Furthermore the hypothesis provides quantitative theoretical support for a catalytic mechanism in which the substrate reacts in its dianionic form and for a mechanism in which the electrophilic attack of the C(4a)-peroxyflavin, or of the hydroxyl radical derived from it, on the benzoate dianion is the rate limiting step in catalysis at pH 8, 25 "C. Finally, it is demonstrated that the hypothesis can be used as a basis for the formulation of working hypotheses in future research, investigating the conversion and regioselective orientation of the various possible substrates in the active site of the wild-type 4-Hydroxybenzoate-3-hydroxylase, its mutants as well as of various other flavin-dependent aromatic hydroxylases, such as for example 3hydroxybenzoate-4-hydroxylase (EC 1.14.13.23), 3-hydroxybenzoate-6-hydroxylase (EC 1.14.13.24) and phenol hydroxylase (EC 1.14.13.7). The flavoprotein 4-Hydroxybenzoate-3-hydroxylase is
