Aerobic benzoyl-CoA catabolic pathway in Azoarcus evansii: studies on the non-oxygenolytic ring cleavage enzyme

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.     

Nonaromatic products from anoxic conversion of benzoyl-CoA with benzoyl-CoA reductase and cyclohexa-1,5-diene-1-carbonyl-CoA hydratase

The enzymes benzoyl-CoA reductase and cyclohex-1, 5-diene-1-carbonyl-CoA hydratase catalyzing the first steps of benzoyl-CoA conversion under anoxic conditions were purified from the denitrifying bacterium, Thauera aromatica. Reaction products obtained with [ring-(13)C(6)]benzoyl-CoA and [ring-(14)C]benzoyl-CoA as substrates were analyzed by high pressure liquid chromatography and by NMR spectroscopy. The main product obtained with titanium(III) citrate or with reduced [8Fe-8S]-ferredoxin from T. aromatica as electron donors was identified as cyclohexa-1, 5-diene-1-carbonyl-CoA. The cyclic diene was converted into 6-hydroxycyclohex-1-ene-1-carbonyl-CoA by the hydratase. Assay mixtures containing reductase, hydratase, and sodium dithionite or a mixture of sulfite and titanium(III) citrate as reducing agent afforded cyclohex-2-ene-1-carbonyl-CoA and 6-hydroxycylohex-2-ene-1-carbonyl-CoA. The potential required for the first electron transfer to the model compound S-ethyl-thiobenzoate yielding a radical anion was determined by cyclic voltammetry as -1.9 V versus a standard hydrogen electrode. The energetics of enzymatic ring reduction of benzoyl-CoA are discussed.     

Genes coding for a new pathway of aerobic benzoate metabolism in Azoarcus evansii

A new pathway for aerobic benzoate oxidation has been postulated for Azoarcus evansii and for a Bacillus stearothermophilus-like strain. Benzoate is first transformed into benzoyl coenzyme A (benzoyl-CoA), which subsequently is oxidized to 3-hydroxyadipyl-CoA and then to 3-ketoadipyl-CoA; all intermediates are CoA thioesters. The genes coding for this benzoate-induced pathway were investigated in the beta-proteobacterium A. evansii. They were identified on the basis of N-terminal amino acid sequences of purified benzoate metabolic enzymes and of benzoate-induced proteins identified on two-dimensional gels. Fifteen genes probably coding for the benzoate pathway were found to be clustered on the chromosome. These genes code for the following functions: a putative ATP-dependent benzoate transport system, benzoate-CoA ligase, a putative benzoyl-CoA oxygenase, a putative isomerizing enzyme, a putative ring-opening enzyme, enzymes for beta-oxidation of CoA-activated intermediates, thioesterase, and lactone hydrolase, as well as completely unknown enzymes belonging to new protein families. An unusual putative regulator protein consists of a regulator protein and a shikimate kinase I-type domain. A deletion mutant with a deletion in one gene (boxA) was unable to grow with benzoate as the sole organic substrate, but it was able to grow with 3-hydroxybenzoate and adipate. The data support the proposed pathway, which postulates operation of a new type of ring-hydroxylating dioxygenase acting on benzoyl-CoA and nonoxygenolytic ring cleavage. A beta-oxidation-like metabolism of the ring cleavage product is thought to lead to 3-ketoadipyl-CoA, which finally is cleaved into succinyl-CoA and acetyl-CoA.     

New aerobic benzoate oxidation pathway via benzoyl-coenzyme A and 3-hydroxybenzoyl-coenzyme A in a denitrifying Pseudomonas sp.

A denitrifying Pseudomonas sp. is able to oxidize aromatic compounds compounds completely to CO2, both aerobically and anaerobically. It is shown that benzoate is aerobically oxidized by a new degradation pathway via benzoyl-coenzyme A (CoA) and 3-hydroxybenzoyl-CoA. The organism grew aerobically with benzoate, 3-hydroxybenzoate, and gentisate; catechol, 2-hydroxybenzoate, and protocatechuate were not used, and 4-hydroxybenzoate was a poor substrate. Mutants were obtained which were not able to utilize benzoate as the sole carbon source aerobically but still used 3-hydroxybenzoate or gentisate. Simultaneous adaptation experiments with whole cells seemingly suggested a sequential induction of enzymes of a benzoate oxidation pathway via 3-hydroxybenzoate and gentisate. Cells grown aerobically with benzoate contained a benzoate-CoA ligase (AMP forming) (0.1 mumol min-1 mg-1) which converted benzoate but not 3-hydroxybenzoate into its CoA thioester. The enzyme of 130 kDa composed of two identical subunits of 56 kDa was purified and characterized. Cells grown aerobically with 3-hydroxybenzoate contained a similarly active CoA ligase for 3-hydroxybenzoate, 3-hydroxybenzoate-CoA ligase (AMP forming). Extracts from cells grown aerobically with benzoate catalyzed a benzoyl-CoA- and flavin adenine dinucleotide-dependent oxidation of NADPH with a specific activity of at least 25 nmol NADPH oxidized min-1 mg of protein-1; NADH and benzoate were not used. This new enzyme, benzoyl-CoA 3-monooxygenase, was specifically induced during aerobic growth with benzoate and converted [U-14C]benzoyl-CoA stoichiometrically to [14C]3-hydroxybenzoyl-CoA.     

Benzoate-coenzyme A ligase from Thauera aromatica: an enzyme acting in anaerobic and aerobic pathways

In the denitrifying member of the beta-Proteobacteria Thauera aromatica, the anaerobic metabolism of aromatic acids such as benzoate or 2-aminobenzoate is initiated by the formation of the coenzyme A (CoA) thioester, benzoyl-CoA and 2-aminobenzoyl-CoA, respectively. Both aromatic substrates were transformed to the acyl-CoA intermediate by a single CoA ligase (AMP forming) that preferentially acted on benzoate. This benzoate-CoA ligase was purified and characterized as a 57-kDa monomeric protein. Based on V(max)/K(m), the specificity constant for 2-aminobenzoate was 15 times lower than that for benzoate; this may be the reason for the slower growth on 2-aminobenzoate. The benzoate-CoA ligase gene was cloned and sequenced and was found not to be part of the gene cluster encoding the general benzoyl-CoA pathway of anaerobic aromatic metabolism. Rather, it was located in a cluster of genes coding for a novel aerobic benzoate oxidation pathway. In line with this finding, the same CoA ligase was induced during aerobic growth with benzoate. A deletion mutant not only was unable to grow anaerobically on benzoate or 2-aminobenzoate, but also aerobic growth on benzoate was affected. This suggests that benzoate induces a single benzoate-CoA ligase. The product of benzoate activation, benzoyl-CoA, then acts as inducer of separate anaerobic or aerobic pathways of benzoyl-CoA, depending on whether oxygen is lacking or present.     

Aerobic benzoyl-coenzyme A (CoA) catabolic pathway in Azoarcus evansii: conversion of ring cleavage product by 3,4-dehydroadipyl-CoA semialdehyde dehydrogenase

Benzoate, a strategic intermediate in aerobic aromatic metabolism, is metabolized in various bacteria via an unorthodox pathway. The intermediates of this pathway are coenzyme A (CoA) thioesters throughout, and ring cleavage is nonoxygenolytic. The fate of the ring cleavage product 3,4-dehydroadipyl-CoA semialdehyde was studied in the beta-proteobacterium Azoarcus evansii. Cell extracts contained a benzoate-induced, NADP(+)-specific aldehyde dehydrogenase, which oxidized this intermediate. A postulated putative long-chain aldehyde dehydrogenase gene, which might encode this new enzyme, is located on a cluster of genes encoding enzymes and a transport system required for aerobic benzoate oxidation. The gene was expressed in Escherichia coli, and the maltose-binding protein-tagged enzyme was purified and studied. It is a homodimer composed of 54 kDa (without tag) subunits and was confirmed to be the desired 3,4-dehydroadipyl-CoA semialdehyde dehydrogenase. The reaction product was identified by nuclear magnetic resonance spectroscopy as the corresponding acid 3,4-dehydroadipyl-CoA. Hence, the intermediates of aerobic benzoyl-CoA catabolic pathway recognized so far are benzoyl-CoA; 2,3-dihydro-2,3-dihydroxybenzoyl-CoA; 3,4-dehydroadipyl-CoA semialdehyde plus formate; and 3,4-dehydroadipyl-CoA. The further metabolism is thought to lead to 3-oxoadipyl-CoA, the intermediate at which the conventional and the unorthodox pathways merge.     

New enzymes involved in aerobic benzoate metabolism in Azoarcus evansii

A new principle of aerobic aromatic metabolism has been postulated, which is in contrast to the known pathways. In various bacteria the aromatic substrate benzoate is first converted to its coenzyme A (CoA) thioester, benzoyl-CoA, which is subsequently attacked by an oxygenase, followed by a non-oxygenolytic fission of the ring. We provide evidence for this hypothesis and show that benzoyl-CoA conversion in the bacterium Azoarcus evansii requires NADPH, O(2) and two protein components, BoxA and BoxB. BoxA is a homodimeric 46 kDa iron-sulphur-flavoprotein, which acts as reductase. In the absence of BoxB, BoxA catalyses the benzoyl-CoA stimulated artificial transfer of electrons from NADPH to O(2) via free FADH(2) to produce H(2)O(2). Physiologically, BoxA uses NADPH to reduce BoxB, a monomeric 55 kDa iron-protein that acts as benzoyl-CoA oxygenase. The product of benzoyl-CoA oxidation was identified by NMR spectroscopy as its dihydrodiol derivative, 2,3-dihydro-2,3-dihydroxybenzoyl-CoA. This suggests that BoxBA act as a benzoyl-CoA dioxygenase/reductase. Unexpectedly, benzoyl-CoA transformation by BoxBA was greatly stimulated when another enoyl-CoA hydratase/isomerase-like protein, BoxC, was added that catalysed the further transformation of the dihydrodiol product formed from benzoyl-CoA. The benzoyl-CoA oxygenase system has very low similarity to known (di)oxygenase systems and is the first member of a new enzyme family.     

Reinvestigation of a new type of aerobic benzoate metabolism in the proteobacterium Azoarcus evansii

The aerobic metabolism of benzoate in the proteobacterium Azoarcus evansii was reinvestigated. The known pathways leading to catechol or protocatechuate do not operate in this bacterium. The presumed degradation via 3-hydroxybenzoyl-coenzyme A (CoA) and gentisate could not be confirmed. The first committed step is the activation of benzoate to benzoyl-CoA by a specifically induced benzoate-CoA ligase (AMP forming). This enzyme was purified and shown to differ from an isoenzyme catalyzing the same reaction under anaerobic conditions. The second step postulated involves the hydroxylation of benzoyl-CoA to a so far unknown product by a novel benzoyl-CoA oxygenase, presumably a multicomponent enzyme system. An iron-sulfur flavoprotein, which may be a component of this system, was purified and characterized. The homodimeric enzyme had a native molecular mass of 98 kDa as determined by gel filtration and contained 0.72 mol flavin adenine dinucleotide (FAD), 10.4 to 18.4 mol of Fe, and 13.3 to 17.9 mol of acid-labile sulfur per mol of native protein, depending on the method of protein determination. This benzoate-induced enzyme catalyzed a benzoyl-CoA-, FAD-, and O2-dependent NADPH oxidation surprisingly without hydroxylation of the aromatic ring; however, H2O2 was formed. The gene (boxA, for benzoate oxidation) coding for this protein was cloned and sequenced. It coded for a protein of 46 kDa with two amino acid consensus sequences for two [4Fe-4S] centers at the N terminus. The deduced amino acid sequence showed homology with subunits of ferredoxin-NADP reductase, nitric oxide synthase, NADPH-cytochrome P450 reductase, and phenol hydroxylase. Upstream of the boxA gene, another gene, boxB, encoding a protein of 55 kDa was found. The boxB gene exhibited homology to open reading frames in various other bacteria which code for components of a putative aerobic phenylacetyl-CoA oxidizing system. The boxB gene product was one of at least five proteins induced when A. evansii was grown on benzoate.     

Functional genomics by NMR spectroscopy. Phenylacetate catabolism in Escherichia coli.

Aerobic metabolism of phenylalanine in most bacteria proceeds via oxidation to phenylacetate. Surprisingly, the further metabolism of phenylacetate has not been elucidated, even in well studied bacteria such as Escherichia coli. The only committed step is the conversion of phenylacetate into phenylacetyl-CoA. The paa operon of E. coli encodes 14 polypeptides involved in the catabolism of phenylacetate. We have found that E. coli K12 mutants with a deletion of the paaF, paaG, paaH, paaJ or paaZ gene are unable to grow with phenylacetate as carbon source. Incubation of a paaG mutant with [U-13C8]phenylacetate yielded ring-1,2-dihydroxy-1,2-dihydrophenylacetyl lactone as shown by NMR spectroscopy. Incubation of the paaF and paaH mutants with phenylacetate yielded delta3-dehydroadipate and 3-hydroxyadipate, respectively. The origin of the carbon atoms of these C6 compounds from the aromatic ring was shown using [ring-13C6]phenylacetate. The paaG and paaZ mutants also converted phenylacetate into ortho-hydroxyphenylacetate, which was previously identified as a dead end product of phenylacetate catabolism. These data, in conjunction with protein sequence data, suggest a novel catabolic pathway via CoA thioesters. According to this, phenylacetyl-CoA is attacked by a ring-oxygenase/reductase (PaaABCDE proteins), generating a hydroxylated and reduced derivative of phenylacetyl-CoA, which is not re-oxidized to a dihydroxylated aromatic intermediate, as in other known aromatic pathways. Rather, it is proposed that this nonaromatic intermediate CoA ester is further metabolized in a complex reaction sequence comprising enoyl-CoA isomerization/hydration, nonoxygenolytic ring opening, and dehydrogenation catalyzed by the PaaG and PaaZ proteins. The subsequent beta-oxidation-type degradation of the resulting CoA dicarboxylate via beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA appears to be catalyzed by the PaaJ, PaaF and PaaH proteins.     

Growth substrate- and phase-specific expression of biphenyl, benzoate, and C1 metabolic pathways in Burkholderia xenovorans LB400

Recent microarray experiments suggested that Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB)-degrading bacterium, utilizes up to three apparently redundant benzoate pathways and a C(1) metabolic pathway during biphenyl and benzoate metabolism. To better characterize the roles of these pathways, we performed quantitative proteome profiling of cells grown on succinate, benzoate, or biphenyl and harvested during either mid-logarithmic growth or the transition between the logarithmic and stationary growth phases. The Bph enzymes, catabolizing biphenyl, were approximately 16-fold more abundant in biphenyl- versus succinate-grown cells. Moreover, the upper and lower bph pathways were independently regulated. Expression of each benzoate pathway depended on growth substrate and phase. Proteins specifying catabolism via benzoate dihydroxylation and catechol ortho-cleavage (ben-cat pathway) were approximately an order of magnitude more abundant in benzoate- versus biphenyl-grown cells at the same growth phase. The chromosomal copy of the benzoyl-coenzyme A (CoA) (box(C)) pathway was also expressed during growth on biphenyl: Box(C) proteins were approximately twice as abundant as Ben and Cat proteins under these conditions. By contrast, proteins of the megaplasmid copy of the benzoyl-CoA (box(M)) pathway were only detected in transition-phase benzoate-grown cells. Other proteins detected at increased levels in benzoate- and biphenyl-grown cells included general stress response proteins potentially induced by reactive oxygen species formed during aerobic aromatic catabolism. Finally, C(1) metabolic enzymes were present in biphenyl-grown cells during transition phase. This study provides insights into the physiological roles and integration of apparently redundant catabolic pathways in large-genome bacteria and establishes a basis for investigating the PCB-degrading abilities of this strain.     

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

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.     

Selenocysteine-containing proteins in anaerobic benzoate metabolism of Desulfococcus multivorans

The sulfate-reducing bacterium Desulfococcus multivorans uses various aromatic compounds as sources of cell carbon and energy. In this work, we studied the initial steps in the aromatic metabolism of this strictly anaerobic model organism. An ATP-dependent benzoate coenzyme A (CoA) ligase (AMP plus PPi forming) composed of a single 59-kDa subunit was purified from extracts of cells grown on benzoate. Specific activity was highest with benzoate and some benzoate derivatives, whereas aliphatic carboxylic acids were virtually unconverted. The N-terminal amino acid sequence showed high similarities with benzoate CoA ligases from Thauera aromatica and Azoarcus evansii. When cultivated on benzoate, cells strictly required selenium and molybdenum, whereas growth on nonaromatic compounds, such as cyclohexanecarboxylate or lactate, did not depend on the presence of the two trace elements. The growth rate on benzoate was half maximal with 1 nM selenite present in the growth medium. In molybdenum- and/or selenium-depleted cultures, growth on benzoate could be induced by addition of the missing trace elements. In extracts of cells grown on benzoate in the presence of [75Se]selenite, three radioactively labeled proteins with molecular masses of approximately 100, 30, and 27 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The 100- and 30-kDa selenoproteins were 5- to 10-fold induced in cells grown on benzoate compared to cells grown on lactate. These results suggest that the dearomatization process in D. multivorans is not catalyzed by the ATP-dependent Fe-S enzyme benzoyl-CoA reductase as in facultative anaerobes but rather involves unknown molybdenum- and selenocysteine-containing proteins.     

Use of benzoate as an electron acceptor by Syntrophus aciditrophicus grown in pure culture with crotonate

In methanogenic environments, the main fate of benzoate is its oxidization to acetate, H(2) and CO(2) by syntrophic associations of hydrogen-producing benzoate degraders and hydrogen-using methanogens. Here, we report the use of benzoate as an electron acceptor. Pure cultures of S. aciditrophicus simultaneously degraded crotonate and benzoate when both substrates were present. The growth rate was 0.007 h(-1) with crotonate and benzoate present compared with 0.025 h(-1) with crotonate alone. After 8 days of incubation, 4.12 +/- 0.50 mM of cyclohexane carboxylate and 8.40 +/- 0.61 mM of acetate were formed and 4.0 +/- 0.04 mM of benzoate and 4.8 +/- 0.5 mM of crotonate were consumed. The molar growth yield was 22.7 +/- 2.1 g (dry wt) of cells per mol of crotonate compared with about 14.0 +/- 0.1 g (dry wt) of cells per mol of crotonate when S. aciditrophicus was grown with crotonate alone. Cultures grown with [ring-(13)C]-benzoate and unlabelled crotonate initially formed [ring-(13)C]-labelled cyclohexane carboxylate. No (13)C-labelled acetate was detected. In addition to cyclohexane carboxylate, (13)C-labelled cyclohex-1-ene carboxylate was detected as an intermediate. Once almost all of the benzoate was gone, carbon isotopic analyses showed that cyclohexane carboxylate was formed from both labelled and non-labelled metabolites. Glutarate and pimelate were also detected at this time and carbon isotopic analyses showed that each was made from a mixture labelled and non-labelled metabolites. The increase in molar growth yield with crotonate and benzoate and the formation of [ring-(13)C]-cyclohexane carboxylate from [ring-(13)C]-benzoate in the presence of crotonate are consistent with benzoate serving as an electron acceptor.     

Metabolism of benzoate, cyclohex-1-ene carboxylate, and cyclohexane carboxylate by "Syntrophus aciditrophicus" strain SB in syntrophic association with H(2)-using microorganisms

The metabolism of benzoate, cyclohex-1-ene carboxylate, and cyclohexane carboxylate by "Syntrophus aciditrophicus" in cocultures with hydrogen-using microorganisms was studied. Cyclohexane carboxylate, cyclohex-1-ene carboxylate, pimelate, and glutarate (or their coenzyme A [CoA] derivatives) transiently accumulated during growth with benzoate. Identification was based on comparison of retention times and mass spectra of trimethylsilyl derivatives to the retention times and mass spectra of authentic chemical standards. (13)C nuclear magnetic resonance spectroscopy confirmed that cyclohexane carboxylate and cyclohex-1-ene carboxylate were produced from [ring-(13)C(6)]benzoate. None of the metabolites mentioned above was detected in non-substrate-amended or heat-killed controls. Cyclohexane carboxylic acid accumulated to a concentration of 260 microM, accounting for about 18% of the initial benzoate added. This compound was not detected in culture extracts of Rhodopseudomonas palustris grown phototrophically or Thauera aromatica grown under nitrate-reducing conditions. Cocultures of "S. aciditrophicus" and Methanospirillum hungatei readily metabolized cyclohexane carboxylate and cyclohex-1-ene carboxylate at a rate slightly faster than the rate of benzoate metabolism. In addition to cyclohexane carboxylate, pimelate, and glutarate, 2-hydroxycyclohexane carboxylate was detected in trace amounts in cocultures grown with cyclohex-1-ene carboxylate. Cyclohex-1-ene carboxylate, pimelate, and glutarate were detected in cocultures grown with cyclohexane carboxylate at levels similar to those found in benzoate-grown cocultures. Cell extracts of "S. aciditrophicus" grown in a coculture with Desulfovibrio sp. strain G11 with benzoate or in a pure culture with crotonate contained the following enzyme activities: an ATP-dependent benzoyl-CoA ligase, cyclohex-1-ene carboxyl-CoA hydratase, and 2-hydroxycyclohexane carboxyl-CoA dehydrogenase, as well as pimelyl-CoA dehydrogenase, glutaryl-CoA dehydrogenase, and the enzymes required for conversion of crotonyl-CoA to acetate. 2-Ketocyclohexane carboxyl-CoA hydrolase activity was detected in cell extracts of "S. aciditrophicus"-Desulfovibrio sp. strain G11 benzoate-grown cocultures but not in crotonate-grown pure cultures of "S. aciditrophicus". These results are consistent with the hypothesis that ring reduction during syntrophic benzoate metabolism involves a four- or six-electron reduction step and that once cyclohex-1-ene carboxyl-CoA is made, it is metabolized in a manner similar to that in R. palustris.     

Identification and characterization of a succinyl-coenzyme A (CoA):benzoate CoA transferase in Geobacter metallireducens

Geobacter metallireducens is a Fe(III)-respiring deltaproteobacterium and serves as a model organism for aromatic compound-degrading, obligately anaerobic bacteria. In this study, a genetic system was established for G. metallireducens using nitrate as an alternative electron acceptor. Surprisingly, disruption of the benzoate-induced bamY gene, encoding a benzoate coenzyme A (CoA) ligase, reproducibly showed an increased biomass yield in comparison to the wild type during growth with benzoate but not during growth with acetate. Complementation of bamY in trans converted the biomass yield back to the wild-type level. Growth of the bamY mutant with benzoate can be rationalized by the identification of a previously unknown succinyl-CoA:benzoate CoA transferase activity; it represents an additional, energetically less demanding mode of benzoate activation. The activity was highly enriched from extracts of cells grown on benzoate, yielding a 50-kDa protein band; mass spectrometric analysis identified the corresponding benzoate-induced gene annotated as a CoA transferase. It was heterologously expressed in Escherichia coli and characterized as a specific succinyl-CoA:benzoate CoA transferase. The newly identified enzyme in conjunction with a benzoate-induced succinyl-CoA synthetase links the tricarboxylic acid cycle to the upper benzoyl-CoA degradation pathway during growth on aromatic growth substrates.     

Benzoyl-coenzyme A thioesterase of Azoarcus evansii: properties and function

The aerobic benzoate metabolism in Azoarcus evansii follows an unusual route. The intermediates of the pathway are processed as coenzyme A (CoA) thioesters and the cleavage of the aromatic ring is non-oxygenolytic. The enzymes of this pathway are encoded by the box gene cluster which harbors a gene, orf1, coding for a putative thioesterase. Benzoyl-CoA thioesterase activity (20 nmol min⁻¹ mg⁻¹ protein) was present in cells grown aerobically on benzoate, but was lacking in cells grown on other aromatic or aliphatic substrates under oxic or anoxic conditions. The gene was cloned and overexpressed in Escherichia coli to produce a C-terminal His-tag fusion protein. The recombinant enzyme was a homotetramer of 16 kDa subunits. It catalyzed not only the hydrolysis of benzoyl-CoA, but also of 2,3-dihydro-2,3-dihydroxybenzoyl-CoA, the second intermediate in the pathway. The enzyme exhibited higher activity with mono-substituted derivatives of benzoyl-CoA, showing highest activity with 4-hydroxybenzoyl-CoA. Di-substituted derivatives of benzoyl-CoA, phenylacetyl-CoA, and aliphatic CoA thioesters were not hydrolyzed but some acted as inhibitors. The thioesterase appears to protect the cell from CoA pool depletion. It may constitute the prototype of a new subfamily within the hotdog fold enzyme superfamily.