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Georg Fuchs - One of the best experts on this subject based on the ideXlab platform.
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Microbial degradation of aromatic compounds — from one strategy to four
Nature Reviews Microbiology, 2011Co-Authors: Georg Fuchs, Matthias Boll, Johann HeiderAbstract:Until recently, it was though that the microbial degradation of aromatic compounds was absolutely dependent on the use of molecular oxygen for ring cleavage. However, as described here by Fuchs and colleagues, microorganisms have other ways of breaking down these compounds when oxygen is scarce or absent. Aromatic compounds are common growth substrates for microorganisms and are also prominent environmental pollutants. The crucial step in their degradation is to overcome the huge resonance energy that stabilizes the aromatic ring. The microbial degradation of aromatic compounds includes a great variety of mechanisms and chemical reactions, some of which have only rare counterparts in organic chemistry. The classical strategy for aromatic-ring cleavage, which is restricted to aerobic organisms, comprises an attack by oxygenases that hydroxylate and finally cleave the ring with the help of activated molecular oxygen (O_2). However, three additional strategies exist that are based on completely different ring activation mechanisms and use CoA thioesters and ring cleavage by hydrolysis. The first strategy depends on the presence of O_2 and uses monooxygenases of the class I di-iron protein family. These enzymes are epoxidases that form non-aromatic ring-epoxides of Benzoyl-CoA and phenylacetyl-CoA, compounds that are further metabolized by ring hydrolysis. The other two strategies are O_2 independent, involve the reduction of the aromatic ring and, under anoxic conditions, convert most aromatic substrates into Benzoyl-CoA as a central intermediate. One of the O_2-independent strategies for aromatic-ring cleavage is used by facultative anaerobic bacteria when they are growing phototrophically or during anaerobic respiration. In these circumstances, these bacteria reduce Benzoyl-CoA by an ATP-driven Benzoyl-CoA reductase that hydrolyses two molecules of ATP. By contrast, during low-energy-yield anaerobic respiration or fermentation, strictly anaerobic bacteria use a different class of Benzoyl-CoA reductase that is ATP independent. The low redox potential that is required for Benzoyl-CoA reduction is probably achieved by a process called electron bifurcation. Electrons from two reduced ferredoxins are split via a flavin cofactor into low- and high-redox-potential electrons. Low-redox-potential electrons may reduce the aromatic ring, whereas high-redox-potential electrons may reduce NAD^+. Aromatic compounds are both common growth substrates for microorganisms and prominent environmental pollutants. The crucial step in their degradation is overcoming the resonance energy that stabilizes the ring structure. The classical strategy for degradation comprises an attack by oxygenases that hydroxylate and finally cleave the ring with the help of activated molecular oxygen. Here, we describe three alternative strategies used by microorganisms to degrade aromatic compounds. All three of these methods involve the use of CoA thioesters and ring cleavage by hydrolysis. However, these strategies are based on different ring activation mechanisms that consist of either formation of a non-aromatic ring-epoxide under oxic conditions, or reduction of the aromatic ring under anoxic conditions using one of two completely different systems.
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Structure and Mechanism of the Diiron Benzoyl-Coenzyme A Epoxidase BoxB.
Journal of Biological Chemistry, 2011Co-Authors: Liv J. Rather, Georg Fuchs, Wael Ismail, Tobias Weinert, Ulrike Demmer, Eckhard Bill, Ulrich ErmlerAbstract:The coenzyme A (CoA)-dependent aerobic benzoate metabolic pathway uses an unprecedented chemical strategy to overcome the high aromatic resonance energy by forming the non-aromatic 2,3-epoxyBenzoyl-CoA. The crucial dearomatizing reaction is catalyzed by three enzymes, BoxABC, where BoxA is an NADPH-dependent reductase, BoxB is a Benzoyl-CoA 2,3-epoxidase, and BoxC is an epoxide ring hydrolase. We characterized the key enzyme BoxB from Azoarcus evansii by structural and Mossbauer spectroscopic methods as a new member of class I diiron enzymes. Several family members were structurally studied with respect to the diiron center architecture, but no structure of an intact diiron enzyme with its natural substrate has been reported. X-ray structures between 1.9 and 2.5 A resolution were determined for BoxB in the diferric state and with bound substrate Benzoyl-CoA in the reduced state. The substrate-bound reduced state is distinguished from the diferric state by increased iron-ligand distances and the absence of directly bridging groups between them. The position of Benzoyl-CoA inside a 20 A long channel and the position of the phenyl ring relative to the diiron center are accurately defined. The C2 and C3 atoms of the phenyl ring are closer to one of the irons. Therefore, one oxygen of activated O(2) must be ligated predominantly to this proximate iron to be in a geometrically suitable position to attack the phenyl ring. Consistent with the observed iron/phenyl geometry, BoxB stereoselectively should form the 2S,3R-epoxide. We postulate a reaction cycle that allows a charge delocalization because of the phenyl ring and the electron-withdrawing CoA thioester.
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Coenzyme A-dependent Aerobic Metabolism of Benzoate via Epoxide Formation
Journal of Biological Chemistry, 2010Co-Authors: Liv J. Rather, Bettina Knapp, Wolfgang Haehnel, Georg FuchsAbstract:In the aerobic metabolism of aromatic substrates, oxygenases use molecular oxygen to hydroxylate and finally cleave the aromatic ring. In the case of the common intermediate benzoate, the ring cleavage substrates are either catechol (in bacteria) or 3,4-dihydroxybenzoate (protocatechuate, mainly in fungi). We have shown before that many bacteria, e.g. Azoarcus evansii, the organism studied here, use a completely different mechanism. This elaborate pathway requires formation of Benzoyl-CoA, followed by an oxygenase reaction and a nonoxygenolytic ring cleavage. Benzoyl-CoA transformation is catalyzed by the iron-containing Benzoyl-CoA oxygenase (BoxB) in conjunction with an FAD and iron-sulfur centers containing reductase (BoxA), which donates electrons from NADPH. Here we show that Benzoyl-CoA oxygenase actually does not form the 2,3-dihydrodiol of Benzoyl-CoA, as formerly postulated, but the 2,3-epoxide. An enoyl-CoA hydratase (BoxC) uses two molecules of water to first hydrolytically open the ring of 2,3-epoxyBenzoyl-CoA, which may proceed via its tautomeric seven-membered oxepin ring form. Then ring C2 is hydrolyzed off as formic acid, yielding 3,4-dehydroadipyl-CoA semialdehyde. The semialdehyde is oxidized by a NADP(+)-dependent aldehyde dehydrogenase (BoxD) to 3,4-dehydroadipyl-CoA. Final products of the pathway are formic acid, acetyl-CoA, and succinyl-CoA. This overlooked pathway occurs in 4-5% of all bacteria whose genomes have been sequenced and represents an elegant strategy to cope with the high resonance energy of aromatic substrates by forming a nonaromatic epoxide.
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Unusual reactions involved in anaerobic metabolism of phenolic compounds.
Biological Chemistry, 2005Co-Authors: Matthias Boll, Georg FuchsAbstract:Aerobic bacteria use molecular oxygen as a common co-substrate for key enzymes of aromatic metabolism. In contrast, in anaerobes all oxygen-dependent reactions are replaced by a set of alternative enzymatic processes. The anaerobic degradation of phenol to a non-aromatic product involves enzymatic processes that are uniquely found in the aromatic metabolism of anaerobic bacteria: (i) ATP-dependent phenol carboxylation to 4-hydroxybenzoate via a phenylphosphate intermediate (biological Kolbe-Schmitt carboxylation); (ii) reductive dehydroxylation of 4-hydroxyBenzoyl-CoA to Benzoyl-CoA; and (iii) ATP-dependent reductive dearomatization of the key intermediate Benzoyl-CoA in a 'Birch-like' reduction mechanism. This review summarizes the results of recent mechanistic studies of the enzymes involved in these three key reactions.
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Aerobic Benzoyl-CoA catabolic pathway in Azoarcus evansii: studies on the non-oxygenolytic ring cleavage enzyme.
Molecular Microbiology, 2005Co-Authors: Johannes Gescher, Wolfgang Eisenreich, Adelbert Bacher, Jürgen Wörth, Georg FuchsAbstract:A novel aerobic benzoate pathway has recently been discovered in various bacteria in which benzoate is first converted to Benzoyl-CoA. The further downstream steps are associated with the gene products of the benzoate oxidation gene cluster (box) on the Azoarcus evansii chromosome. Benzoyl-CoA is oxidized to 2,3-dihydro-2,3-dihydroxyBenzoyl-CoA (Benzoyl-CoA dihydrodiol) by Benzoyl-CoA oxygenase/reductase BoxBA in the presence of molecular oxygen. This study identified the next, ring cleaving step catalysed by BoxC. The boxC gene was expressed in a recombinant Escherichia coli strain as a fusion protein with maltose binding protein (BoxC(mal)) and the wild type as well as the recombinant proteins were purified and studied. BoxC catalyses the reaction 2,3-dihydro-2,3-dihydroxyBenzoyl-CoA + H(2)O --> 3,4-dehydroadipyl-CoA semialdehyde + HCOOH. This is supported by the following results. Assays containing [ring-(13)C(6)]Benzoyl-CoA, Benzoyl-CoA oxygenase/reductase, BoxC(mal) protein, NADPH and semicarbazide were analysed directly by NMR spectroscopy and mass spectrometry. The products were identified as the semicarbazone of [2,3,4,5,6-(13)C(5)]3,4-dehydroadipyl-CoA semialdehyde; the missing one-carbon unit being formate. The same reaction mixture without semicarbazide yielded a mixture of the hydrate of [2,3,4,5,6-(13)C(5)]3,4-dehydroadipyl-CoA semialdehyde and [2,3,4,5,6-(13)C(5)]4,5-dehydroadipyl-CoA semialdehyde. BoxC, a 122 kDa homodimeric enzyme (61 kDa subunits), is termed Benzoyl-CoA-dihydrodiol lyase. It contains domains characteristic for enoyl-CoA hydratases/isomerases, besides a large central domain with no significant similarity to sequences in the database. The purified protein did not require divalent metals, molecular oxygen or any cosubstrates or coenzymes for activity. The complex reaction is part of a widely distributed new principle of aerobic aromatic metabolism in which all intermediates are coenzyme A thioesters and the actual ring-cleavage reaction does not require molecular oxygen.
Matthias Boll - One of the best experts on this subject based on the ideXlab platform.
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An Aerobic Hybrid Phthalate Degradation Pathway via Phthaloyl-Coenzyme A in Denitrifying Bacteria
Applied and Environmental Microbiology, 2020Co-Authors: Christa Ebenau-jehle, Christina I. S. L. Soon, Jonathan Fuchs, Robin Geiger, Matthias BollAbstract:ABSTRACT The degradation of the xenobiotic phthalic acid esters by microorganisms is initiated by the hydrolysis to the respective alcohols and ortho-phthalate (hereafter, phthalate). In aerobic bacteria and fungi, oxygenases are involved in the conversion of phthalate to protocatechuate, the substrate for ring-cleaving dioxygenases. In contrast, anaerobic bacteria activate phthalate to the extremely unstable phthaloyl-coenzyme A (CoA), which is decarboxylated by oxygen-sensitive UbiD-like phthaloyl-CoA decarboxylase (PCD) to the central Benzoyl-CoA intermediate. Here, we demonstrate that the facultatively anaerobic, denitrifying Thauera chlorobenzoica 3CB-1 and Aromatoleum evansii KB740 strains use phthalate as a growth substrate under aerobic and denitrifying conditions. In vitro assays with extracts from cells grown aerobically with phthalate demonstrated the succinyl-CoA-dependent activation of phthalate followed by decarboxylation to Benzoyl-CoA. In T. chlorobenzoica 3CB-1, we identified PCD as a highly abundant enzyme in both aerobically and anaerobically grown cells, whereas genes for phthalate dioxygenases are missing in the genome. PCD was highly enriched from aerobically grown T. chlorobenzoica cells and was identified as an identical enzyme produced under denitrifying conditions. These results indicate that the initial steps of aerobic phthalate degradation in denitrifying bacteria are accomplished by the anaerobic enzyme inventory, whereas the Benzoyl-CoA oxygenase-dependent pathway is used for further conversion to central intermediates. Such a hybrid pathway requires intracellular oxygen homeostasis at concentrations low enough to prevent PCD inactivation but sufficiently high to supply Benzoyl-CoA oxygenase with its cosubstrate. IMPORTANCE Phthalic acid esters (PAEs) are industrially produced on a million-ton scale per year and are predominantly used as plasticizers. They are classified as environmentally relevant xenobiotics with a number of adverse health effects, including endocrine-disrupting activity. Biodegradation by microorganisms is considered the most effective process to eliminate PAEs from the environment. It is usually initiated by the hydrolysis of PAEs to alcohols and o-phthalic acid. Degradation of o-phthalic acid fundamentally differs in aerobic and anaerobic microorganisms; aerobic phthalate degradation heavily depends on dioxygenase-dependent reactions, whereas anaerobic degradation employs the oxygen-sensitive key enzyme phthaloyl-CoA decarboxylase. We demonstrate that aerobic phthalate degradation in facultatively anaerobic bacteria proceeds via a previously unknown hybrid degradation pathway involving oxygen-sensitive and oxygen-dependent key enzymes. Such a strategy is essential for facultatively anaerobic bacteria that frequently switch between oxic and anoxic environments.
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Breaking Benzene Aromaticity-Computational Insights into the Mechanism of the Tungsten-Containing Benzoyl-CoA Reductase.
Journal of the American Chemical Society, 2017Co-Authors: Martin Culka, Matthias Boll, Simona G Huwiler, G. Matthias UllmannAbstract:Aromatic compounds are environmental pollutants with toxic and carcinogenic properties. Despite the stability of aromatic rings, bacteria are able to degrade the aromatic compounds into simple metabolites and use them as growth substrates under oxic or even under anoxic conditions. In anaerobic microorganisms, most monocyclic aromatic growth substrates are converted to the central intermediate benzoyl-coenzyme A, which is enzymatically reduced to cyclohexa-1,5-dienoyl-CoA. The strictly anaerobic bacterium Geobacter metallireducens uses the class II Benzoyl-CoA reductase complex for this reaction. The catalytic BamB subunit of this complex harbors an active site tungsten-bis-pyranopterin cofactor with the metal being coordinated by five protein/cofactor-derived sulfur atoms and a sixth, so far unknown, ligand. Although BamB has been biochemically and structurally characterized, its mechanism still remains elusive. Here we use continuum electrostatic and QM/MM calculations to model Benzoyl-CoA reduction by ...
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Enzymes of the benzoyl-coenzyme A degradation pathway in the hyperthermophilic archaeon Ferroglobus placidus.
Environmental Microbiology, 2015Co-Authors: Georg Schmid, Sandra Bosch René, Matthias BollAbstract:The Fe(III)-respiring Ferroglobus placidus is the only known archaeon and hyperthermophile for which a complete degradation of aromatic substrates to CO2 has been reported. Recent genome and transcriptome analyses proposed a benzoyl-coenzyme A (CoA) degradation pathway similar to that found in the phototrophic Rhodopseudomonas palustris, which involves a cyclohex-1-ene-1-carboxyl-CoA (1-enoyl-CoA) forming, ATP-dependent key enzyme Benzoyl-CoA reductase (BCR). In this work, we demonstrate, by first in vitro studies, that Benzoyl-CoA is ATP-dependently reduced by two electrons to cyclohexa-1,5-dienoyl-CoA (1,5-dienoyl-CoA), which is further degraded by hydration to 6-hydroxycyclohex-1-ene-1-carboxyl-CoA (6-OH-1-enoyl-CoA); upon addition of NAD(+) , the latter was subsequently converted to β-oxidation intermediates. The four candidate genes of BCR were heterologously expressed, and the enriched, oxygen-sensitive enzyme catalysed the two-electron reduction of Benzoyl-CoA to 1,5-dienoyl-CoA. A gene previously assigned to a 2,3-didehydropimeloyl-CoA hydratase was heterologously expressed and shown to act as a typical 1,5-dienoyl-CoA hydratase that does not accept 1-enoyl-CoA. A gene previously assigned to a 1-enoyl-CoA hydratase was heterologously expressed and identified to code for a bifunctional crotonase/3-OH-butyryl-CoA dehydrogenase. In summary, the results consistently provide biochemical evidence that F. placidus and probably other archaea predominantly degrade aromatics via the Thauera/Azoarcus type and not or only to a minor extent via the predicted R. palustris-type Benzoyl-CoA degradation pathway.
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cyclohexanecarboxyl coenzyme a coa and cyclohex 1 ene 1 carboxyl coa dehydrogenases two enzymes involved in the fermentation of benzoate and crotonate in syntrophus aciditrophicus
Journal of Bacteriology, 2013Co-Authors: Johannes W Kung, Jana Seifert, Martin Von Bergen, Matthias BollAbstract:The strictly anaerobic Syntrophus aciditrophicus is a fermenting deltaproteobacterium that is able to degrade benzoate or crotonate in the presence and in the absence of a hydrogen-consuming partner. During growth in pure culture, both substrates are dismutated to acetate and cyclohexane carboxylate. In this work, the unknown enzymes involved in the late steps of cyclohexane carboxylate formation were studied. Using enzyme assays monitoring the oxidative direction, a cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA)-forming cyclohexanecarboxyl-CoA (ChCoA) dehydrogenase was purified and characterized from S. aciditrophicus and after heterologous expression of its gene in Escherichia coli. In addition, a cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA)-forming Ch1CoA dehydrogenase was characterized after purification of the heterologously expressed gene. Both enzymes had a native molecular mass of 150 kDa and were composed of a single, 40- to 45-kDa subunit; both contained flavin adenine dinucleotide (FAD) as a cofactor. While the ChCoA dehydrogenase was competitively inhibited by Ch1CoA in the oxidative direction, Ch1CoA dehydrogenase further converted the product Ch1,5CoA to Benzoyl-CoA. The results obtained suggest that Ch1,5CoA is a common intermediate in benzoate and crotonate fermentation that serves as an electron-accepting substrate for the two consecutively operating acyl-CoA dehydrogenases characterized in this work. In the case of benzoate fermentation, Ch1,5CoA is formed by a class II Benzoyl-CoA reductase; in the case of crotonate fermentation, Ch1,5CoA is formed by reversing the reactions of the Benzoyl-CoA degradation pathway that are also employed during the oxidative (degradative) branch of benzoate fermentation.
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cyclohexanecarboxyl coenzyme a coa and cyclohex 1 ene 1 carboxyl coa dehydrogenases two enzymes involved in the fermentation of benzoate and crotonate in syntrophus aciditrophicus
Journal of Bacteriology, 2013Co-Authors: Johannes W Kung, Jana Seifert, Martin Von Bergen, Matthias BollAbstract:The strictly anaerobic Syntrophus aciditrophicus is a fermenting deltaproteobacterium that is able to degrade benzoate or crotonate in the presence and in the absence of a hydrogen-consuming partner. During growth in pure culture, both substrates are dismutated to acetate and cyclohexane carboxylate. In this work, the unknown enzymes involved in the late steps of cyclohexane carboxylate formation were studied. Using enzyme assays monitoring the oxidative direction, a cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA)-forming cyclohexanecarboxyl-CoA (ChCoA) dehydrogenase was purified and characterized from S. aciditrophicus and after heterologous expression of its gene in Escherichia coli. In addition, a cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA)-forming Ch1CoA dehydrogenase was characterized after purification of the heterologously expressed gene. Both enzymes had a native molecular mass of 150 kDa and were composed of a single, 40- to 45-kDa subunit; both contained flavin adenine dinucleotide (FAD) as a cofactor. While the ChCoA dehydrogenase was competitively inhibited by Ch1CoA in the oxidative direction, Ch1CoA dehydrogenase further converted the product Ch1,5CoA to Benzoyl-CoA. The results obtained suggest that Ch1,5CoA is a common intermediate in benzoate and crotonate fermentation that serves as an electron-accepting substrate for the two consecutively operating acyl-CoA dehydrogenases characterized in this work. In the case of benzoate fermentation, Ch1,5CoA is formed by a class II Benzoyl-CoA reductase; in the case of crotonate fermentation, Ch1,5CoA is formed by reversing the reactions of the Benzoyl-CoA degradation pathway that are also employed during the oxidative (degradative) branch of benzoate fermentation.
Johannes W Kung - One of the best experts on this subject based on the ideXlab platform.
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Structural basis of enzymatic benzene ring reduction
Nature Chemical Biology, 2015Co-Authors: Tobias Weinert, Johannes W Kung, Simona G Huwiler, Sina Weidenweber, Petra Hellwig, Hans-joachim Stärk, Till Biskup, Stefan Weber, Julien J. H. Cotelesage, Graham N. GeorgeAbstract:Structural, spectroscopic and kinetic analyses suggest that class II Benzoyl-CoA reductases from anaerobic bacteria use an unusual tungsten cofactor and a conserved histidine to perform a reduction akin to the widely used Birch reduction in organic chemistry.
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cyclohexanecarboxyl coenzyme a coa and cyclohex 1 ene 1 carboxyl coa dehydrogenases two enzymes involved in the fermentation of benzoate and crotonate in syntrophus aciditrophicus
Journal of Bacteriology, 2013Co-Authors: Johannes W Kung, Jana Seifert, Martin Von Bergen, Matthias BollAbstract:The strictly anaerobic Syntrophus aciditrophicus is a fermenting deltaproteobacterium that is able to degrade benzoate or crotonate in the presence and in the absence of a hydrogen-consuming partner. During growth in pure culture, both substrates are dismutated to acetate and cyclohexane carboxylate. In this work, the unknown enzymes involved in the late steps of cyclohexane carboxylate formation were studied. Using enzyme assays monitoring the oxidative direction, a cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA)-forming cyclohexanecarboxyl-CoA (ChCoA) dehydrogenase was purified and characterized from S. aciditrophicus and after heterologous expression of its gene in Escherichia coli. In addition, a cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA)-forming Ch1CoA dehydrogenase was characterized after purification of the heterologously expressed gene. Both enzymes had a native molecular mass of 150 kDa and were composed of a single, 40- to 45-kDa subunit; both contained flavin adenine dinucleotide (FAD) as a cofactor. While the ChCoA dehydrogenase was competitively inhibited by Ch1CoA in the oxidative direction, Ch1CoA dehydrogenase further converted the product Ch1,5CoA to Benzoyl-CoA. The results obtained suggest that Ch1,5CoA is a common intermediate in benzoate and crotonate fermentation that serves as an electron-accepting substrate for the two consecutively operating acyl-CoA dehydrogenases characterized in this work. In the case of benzoate fermentation, Ch1,5CoA is formed by a class II Benzoyl-CoA reductase; in the case of crotonate fermentation, Ch1,5CoA is formed by reversing the reactions of the Benzoyl-CoA degradation pathway that are also employed during the oxidative (degradative) branch of benzoate fermentation.
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cyclohexanecarboxyl coenzyme a coa and cyclohex 1 ene 1 carboxyl coa dehydrogenases two enzymes involved in the fermentation of benzoate and crotonate in syntrophus aciditrophicus
Journal of Bacteriology, 2013Co-Authors: Johannes W Kung, Jana Seifert, Martin Von Bergen, Matthias BollAbstract:The strictly anaerobic Syntrophus aciditrophicus is a fermenting deltaproteobacterium that is able to degrade benzoate or crotonate in the presence and in the absence of a hydrogen-consuming partner. During growth in pure culture, both substrates are dismutated to acetate and cyclohexane carboxylate. In this work, the unknown enzymes involved in the late steps of cyclohexane carboxylate formation were studied. Using enzyme assays monitoring the oxidative direction, a cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA)-forming cyclohexanecarboxyl-CoA (ChCoA) dehydrogenase was purified and characterized from S. aciditrophicus and after heterologous expression of its gene in Escherichia coli. In addition, a cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA)-forming Ch1CoA dehydrogenase was characterized after purification of the heterologously expressed gene. Both enzymes had a native molecular mass of 150 kDa and were composed of a single, 40- to 45-kDa subunit; both contained flavin adenine dinucleotide (FAD) as a cofactor. While the ChCoA dehydrogenase was competitively inhibited by Ch1CoA in the oxidative direction, Ch1CoA dehydrogenase further converted the product Ch1,5CoA to Benzoyl-CoA. The results obtained suggest that Ch1,5CoA is a common intermediate in benzoate and crotonate fermentation that serves as an electron-accepting substrate for the two consecutively operating acyl-CoA dehydrogenases characterized in this work. In the case of benzoate fermentation, Ch1,5CoA is formed by a class II Benzoyl-CoA reductase; in the case of crotonate fermentation, Ch1,5CoA is formed by reversing the reactions of the Benzoyl-CoA degradation pathway that are also employed during the oxidative (degradative) branch of benzoate fermentation.
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reversible biological birch reduction at an extremely low redox potential
Journal of the American Chemical Society, 2010Co-Authors: Johannes W Kung, Martin Von Bergen, Michael Müller, Sven Baumann, Peterleon Hagedoorn, Wilfred R Hagen, Matthias BollAbstract:The Birch reduction of aromatic rings to cyclohexadiene compounds is widely used in chemical synthesis and requires solvated electrons, the most potent reductants known in organic chemistry. Benzoyl-coenzyme A (CoA) reductases (BCR) are key enzymes in the anaerobic bacterial degradation of aromatic compounds and catalyze an analogous reaction under physiological conditions. Class I BCRs are FeS enzymes and couple the reductive dearomatization of Benzoyl-CoA to cyclohexa-1,5-diene-1-carboxyl-CoA (dienoyl-CoA) to a stoichiometric ATP hydrolysis. Here, we report on a tungsten-containing class II BCR from Geobacter metallireducens that catalyzed the fully reversible, ATP-independent dearomatization of Benzoyl-CoA to dienoyl-CoA. BCR additionally catalyzed the disproportionation of dienoyl-CoA to Benzoyl-CoA/monoenoyl-CoA and the four- and six-electron reduction of Benzoyl-CoA in the presence of a reduced low-potential bridged 2,2′-bipyridyl redox dye. Reversible redox titration experiments in the presence of ...
Matthias Oll - One of the best experts on this subject based on the ideXlab platform.
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aromatizing cyclohexa 1 5 diene 1 carbonyl coenzyme a oxidase characterization and its role in anaerobic aromatic metabolism
Journal of Biological Chemistry, 2008Co-Authors: Arbel Thiele, Olive Riede, Nico Jehmlich, Marti Von Berge, Michael Mulle, Matthias OllAbstract:Benzoyl-CoA reductases (BCRs) are key enzymes of anaerobic aromatic metabolism in facultatively anaerobic bacteria. The highly oxygen-sensitive enzymes catalyze the ATP-dependent reductive dearomatization of the substrate, yielding cyclohexa-1,5-diene-1-carbonyl-CoA (1,5-dienoyl-CoA). In extracts from anaerobically grown denitrifying Thauera aromatica, we detected a benzoate-induced, Benzoyl-CoA-forming, 1,5-dienoyl-CoA:acceptor oxidoreductase activity. This activity co-purified with BCR but could be partially separated from it by hydroxyapatite chromatography. After activity staining on native gels, a monomeric protein with a subunit molecular weight of Mr 76,000 was identified. Mass spectrometric analysis of tryptic digests identified peptides from NADH oxidases/2,4-dienoyl-CoA reductases/“old yellow” enzymes. The UV-visible spectrum of the enriched enzyme suggested the presence of flavin and Fe/S-cofactors, and it was bleached upon the addition of 1,5-dienoyl-CoA. The enzyme had a high affinity for dioxygen as electron acceptor (Km = 10 μm) and therefore is referred to as 1,5-dienoyl-CoA oxidase (DCO). The likely product formed from dioxygen reduction was H2O. DCO was highly specific for 1,5-dienoyl-CoA (Km = 27 μm). The initial rate of DCO followed a Nernst curve with half-maximal activity at +10 mV. We propose that DCO provides protection for the extremely oxygen-sensitive BCR enzyme when the bacterium degrades aromatic compounds at the edge of steep oxygen gradients. The redox-dependent switch in DCO guarantees that DCO is only active during oxidative stress and circumvents futile dearomatization/rearomatization reactions catalyzed by BCR and DCO.
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benzoyl coenzyme a reductase dearomatizing a key enzyme of anaerobic aromatic metabolism atp dependence of the reaction purification and some properties of the enzyme from thauera aromatica strain k172
FEBS Journal, 1995Co-Authors: Matthias Oll, Georg FuchsAbstract:Anoxic metabolism of many aromatic compounds proceeds via the common intermediate Benzoyl-CoA. Benzoyl-CoA is dearomatized by Benzoyl-CoA reductase (dearomatizing) in a two-electron reduction step, possibly yielding cyclohex-1,5-diene-1-carboxyl-CoA. This process has to overcome a high activation energy and is considered a biological Birch reduction. The central, aromatic-ring-reducing enzyme was investigated for the first time in the denitrifying bacterium Thauera aromatica strain K172. A spectrophotometric assay was developed which was strictly dependent on MgATP, both with cell extract and with purified enzyme. The oxygen-sensitive new enzyme was purified 35-fold with 20% yield under anaerobic conditions in the presence of 0.25 mM dithionite. It had a native molecular mass of approximately 170 kDa and consisted of four subunits a,b,c,d of 48, 45, 38 and 32 kDa. The oligomer composition of the protein most likely is abcd. The ultraviolet/visible spectrum of the protein as isolated, but without dithionite, was characteristic for an iron-sulfur protein with an absorption maximum at 279 nm and a broad shoulder at 390 nm. The estimated molar absorption coefficient at 390 nm was 35,000 M-1 cm-1. Reduction of the enzyme by dithionite resulted in a decrease of absorbance at 390 nm, and the colour turned from greenish-brown to red-brown. The enzyme contained 10.8 +/- 1.5 mol Fe and 10.5 +/- 1.5 mol acid-labile sulfur/mol. Besides zinc (0.5 mol/mol protein) no other metals nor selenium could be detected in significant amounts. The enzyme preparation contained a flavin or flavin-like compound; the estimated content was 0.3 mol/mol enzyme. The enzyme reaction required MgATP and a strong reductant such as Ti(III). The reaction catalyzed is: Benzoyl-CoA + 2 Ti(III) + n ATP-->non-aromatic acyl-CoA + 2 Ti(IV) + n ADP + n Pi. The estimated number n of ATP molecules hydrolyzed/two electrons transferred in Benzoyl-CoA reduction is 2-4. In the absence of Benzoyl-CoA the enzyme exhibited oxygen-sensitive ATPase activity. The enzyme was specific for Mg(2+)-ATP, other nucleoside triphosphates being inactive (< 1%). Mg2+ could be substituted to some extent by Mn2+, Fe2+ and less efficiently by Co2+. Benzoate was not reduced, whereas some fluoro, hydroxy, amino and methyl analogues of the activated benzoic acid were reduced, albeit at much lower rate; the products remain to be identified. The specific activity with reduced methyl viologen as the electron donor was 0.55 mumol min-1 mg-1 corresponding to a catalytic number of 1.6 s-1. The apparent Km values under the assay conditions (0.5 mM for both reduced and oxidized methyl viologen) of Benzoyl-CoA and ATP were 15 microM and 0.6 mM, respectively. The enzyme was inactivated by ethylene, bipyridyl and, in higher concentrations, by acetylene. Benzoyl-CoA reductase also catalyzed the ATP-dependent two-electron reduction of hydroxylamine (Km 0.15 mM) and azide. Some of the properties of the enzyme are reminiscent of those of nitrogenase which similarly overcomes the high activation energy for dinitrogen reduction by coupling electron transfer to the hydrolysis of ATP.
Martin Von Bergen - One of the best experts on this subject based on the ideXlab platform.
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cyclohexanecarboxyl coenzyme a coa and cyclohex 1 ene 1 carboxyl coa dehydrogenases two enzymes involved in the fermentation of benzoate and crotonate in syntrophus aciditrophicus
Journal of Bacteriology, 2013Co-Authors: Johannes W Kung, Jana Seifert, Martin Von Bergen, Matthias BollAbstract:The strictly anaerobic Syntrophus aciditrophicus is a fermenting deltaproteobacterium that is able to degrade benzoate or crotonate in the presence and in the absence of a hydrogen-consuming partner. During growth in pure culture, both substrates are dismutated to acetate and cyclohexane carboxylate. In this work, the unknown enzymes involved in the late steps of cyclohexane carboxylate formation were studied. Using enzyme assays monitoring the oxidative direction, a cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA)-forming cyclohexanecarboxyl-CoA (ChCoA) dehydrogenase was purified and characterized from S. aciditrophicus and after heterologous expression of its gene in Escherichia coli. In addition, a cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA)-forming Ch1CoA dehydrogenase was characterized after purification of the heterologously expressed gene. Both enzymes had a native molecular mass of 150 kDa and were composed of a single, 40- to 45-kDa subunit; both contained flavin adenine dinucleotide (FAD) as a cofactor. While the ChCoA dehydrogenase was competitively inhibited by Ch1CoA in the oxidative direction, Ch1CoA dehydrogenase further converted the product Ch1,5CoA to Benzoyl-CoA. The results obtained suggest that Ch1,5CoA is a common intermediate in benzoate and crotonate fermentation that serves as an electron-accepting substrate for the two consecutively operating acyl-CoA dehydrogenases characterized in this work. In the case of benzoate fermentation, Ch1,5CoA is formed by a class II Benzoyl-CoA reductase; in the case of crotonate fermentation, Ch1,5CoA is formed by reversing the reactions of the Benzoyl-CoA degradation pathway that are also employed during the oxidative (degradative) branch of benzoate fermentation.
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cyclohexanecarboxyl coenzyme a coa and cyclohex 1 ene 1 carboxyl coa dehydrogenases two enzymes involved in the fermentation of benzoate and crotonate in syntrophus aciditrophicus
Journal of Bacteriology, 2013Co-Authors: Johannes W Kung, Jana Seifert, Martin Von Bergen, Matthias BollAbstract:The strictly anaerobic Syntrophus aciditrophicus is a fermenting deltaproteobacterium that is able to degrade benzoate or crotonate in the presence and in the absence of a hydrogen-consuming partner. During growth in pure culture, both substrates are dismutated to acetate and cyclohexane carboxylate. In this work, the unknown enzymes involved in the late steps of cyclohexane carboxylate formation were studied. Using enzyme assays monitoring the oxidative direction, a cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA)-forming cyclohexanecarboxyl-CoA (ChCoA) dehydrogenase was purified and characterized from S. aciditrophicus and after heterologous expression of its gene in Escherichia coli. In addition, a cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA)-forming Ch1CoA dehydrogenase was characterized after purification of the heterologously expressed gene. Both enzymes had a native molecular mass of 150 kDa and were composed of a single, 40- to 45-kDa subunit; both contained flavin adenine dinucleotide (FAD) as a cofactor. While the ChCoA dehydrogenase was competitively inhibited by Ch1CoA in the oxidative direction, Ch1CoA dehydrogenase further converted the product Ch1,5CoA to Benzoyl-CoA. The results obtained suggest that Ch1,5CoA is a common intermediate in benzoate and crotonate fermentation that serves as an electron-accepting substrate for the two consecutively operating acyl-CoA dehydrogenases characterized in this work. In the case of benzoate fermentation, Ch1,5CoA is formed by a class II Benzoyl-CoA reductase; in the case of crotonate fermentation, Ch1,5CoA is formed by reversing the reactions of the Benzoyl-CoA degradation pathway that are also employed during the oxidative (degradative) branch of benzoate fermentation.
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reversible biological birch reduction at an extremely low redox potential
Journal of the American Chemical Society, 2010Co-Authors: Johannes W Kung, Martin Von Bergen, Michael Müller, Sven Baumann, Peterleon Hagedoorn, Wilfred R Hagen, Matthias BollAbstract:The Birch reduction of aromatic rings to cyclohexadiene compounds is widely used in chemical synthesis and requires solvated electrons, the most potent reductants known in organic chemistry. Benzoyl-coenzyme A (CoA) reductases (BCR) are key enzymes in the anaerobic bacterial degradation of aromatic compounds and catalyze an analogous reaction under physiological conditions. Class I BCRs are FeS enzymes and couple the reductive dearomatization of Benzoyl-CoA to cyclohexa-1,5-diene-1-carboxyl-CoA (dienoyl-CoA) to a stoichiometric ATP hydrolysis. Here, we report on a tungsten-containing class II BCR from Geobacter metallireducens that catalyzed the fully reversible, ATP-independent dearomatization of Benzoyl-CoA to dienoyl-CoA. BCR additionally catalyzed the disproportionation of dienoyl-CoA to Benzoyl-CoA/monoenoyl-CoA and the four- and six-electron reduction of Benzoyl-CoA in the presence of a reduced low-potential bridged 2,2′-bipyridyl redox dye. Reversible redox titration experiments in the presence of ...
