Benzoate Oxidation Research Articles

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.

Biomass characteristics in three sequencing batch reactors treating a wastewater containing synthetic organic chemicals

The physical and biochemical characteristics of the biomass in three lab-scale sequencing batch reactors (SBR) treating a synthetic wastewater at a 20-day target solids retention time (SRT) were investigated. The synthetic wastewater feed contained biogenic compounds and 22 organic priming compounds, chosen to represent a wide variety of chemical structures with different N, P and S functional groups. At a two-day hydraulic retention time (HRT), the oxidation-reduction potential (ORP) cycled between -100 (anoxic) and 100 mV (aerobic) in the anoxic/aerobic SBR, while it remained in a range of 126+/-18 and 249+/-18 mV in the aerobic sequencing batch biofilm reactor (SBBR) and the aerobic SBR reactor, respectively. A granular activated sludge with excellent settleability (SVI=98+/-31 L mg(-1)) developed only in the anoxic/aerobic SBR, compared to a bulky sludge with poor settling characteristics in the aerobic SBR and SBBR. While all reactors had very good COD removal (>90%) and displayed nitrification, substantial nitrogen removal (74%) was only achieved in the anoxic/aerobic SBR. During the entire operational period, benzoate, theophylline and 4-chlorophenol were completely removed in all reactors. In contrast, effluent 3-nitrobenzoate was recorded when its influent concentration was increased to 5 mg L(-1) and dropped only to below 1 mg L(-1) after 300 days of operation. The competent (active) biomass fractions for these compounds were between 0.04% and 5.52% of the total biomass inferred from substrate-specific microbial enumerations. The measured competent biomass fractions for 4-chlorophenol and 3-nitrobenzoate degradation were significantly lower than the influent COD fractions of these compounds. Correspondent to the highest competent biomass fraction for benzoate degradation among the test SOCs, benzoate oxidation could be quantified with an extant respirometric technique, with the highest specific oxygen uptake rate (SOUR(benzoate), 0.026 g O2 h(-1) g(-1) XCOD) in the anoxic/aerobic SBR. These combined results suggest that operating SBRs with alternative anoxic/aerobic cycles might facilitate the formation of granular sludge with good settleability, and retain comparable removal of nitrogen and synthetic organic compounds. Hence, the practice of anoxic/aerobic cycling should be considered in wastewater treatment systems whenever possible.

Oxidation Potentials of Soil Organic Matter in Histosols under Different Tillage Methods

Organic soils in the Everglades Agricultural Area (EAA) of southern Florida, USA, are subsiding due primarily to oxidation by aerobic microorganisms. Since there are large C losses in drained Histosols, it is not certain if minimum tillage will significantly reduce soil C losses below the background levels. The objective of this experiment was to determine if increasing the surface soil disturbance through tillage significantly increases the soil organic matter oxidation potential (OP) in Histosols. Tillage treatments from lowest to highest soil disturbance consisted of (i) no‐till; (ii) tine implement cultivation, one pass; (iii) tine implement cultivation, two passes; (iv) heavy harrow disking; and (v) switchplowing. Treatments were conducted on two fields (bare fallow and plant residue covered). Surface soil samples (0–15 cm) and all microbial measurements were taken on 0, 1, 4, 13, 28, and 42 d after tillage. The switchplow treatment had the greatest OP (14C oxidation of benzoate) and soil microbial CO2 respiration (RESP) averaged across the 42‐d period compared with the other treatments. No‐till tended to have the lowest OP and RESP. Other tillage treatment effects were intermediate depending on field type. Correlation analyses indicated that greater quantities of extractable N (EXN) were related to increased OP in both fields, while higher quantities of extractable C (EXC) were related to increased RESP in both fields. This research demonstrates that minimum tillage can be used on Histosols to reduce C loss and thereby reduce soil subsidence.

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.

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.

Benzoate 1,2-dioxygenase from Pseudomonas putida: single turnover kinetics and regulation of a two-component Rieske dioxygenase.

The benzoate 1,2-dioxygenase system (BZDOS) from Pseudomonas putida mt-2 catalyzes the NADH-dependent oxidation of benzoate to 1-carboxy-1,2-cis-dihydroxycyclohexa-3,5-diene. Both the oxygenase (BZDO) and reductase (BZDR) components of BZDOS have been purified and characterized kinetically and by optical, EPR, and Mössbauer spectroscopies. BZDO has an (alpha beta)(3) subunit structure in which each alpha subunit contains a Rieske [2Fe-2S] cluster and a mononuclear iron site. Two different purification protocols were developed for BZDO allowing the mononuclear iron to be stabilized in either the Fe(III) or the Fe(II) state for spectroscopic characterization. Using single turnover reactions, it is shown that fully reduced BZDO alone is capable of yielding the cis-diol product in high yield at rates that exceed the BZDOS turnover number. At the conclusion of turnover, quantification of each oxidation state of the metal sites by EPR and Mössbauer spectroscopies shows that the Rieske cluster and mononuclear iron are each oxidized in amounts equal to the product yield, suggesting that the two electrons required for catalysis derive from the two metal centers. These results are in agreement with our previous study of naphthalene 1,2-dioxygenase [Wolfe, M. D., Parales, J. V., Gibson, D. T., and Lipscomb, J. D. (2001) J. Biol. Chem. 276, 1945-1953], which belongs to a different Rieske dioxygenase subclass, suggesting that it is a universal characteristic of Rieske dioxygenases that oxygen activation and substrate oxidation are catalyzed by the oxygenase component alone. The EPR spectrum of the Fe(III) center after a single turnover is distinct from either of those of substrate-free or substrate-bound enzyme. The complex with this spectrum is not formed by addition of cis-diol product to the resting Fe(III) form of the enzyme but is observed when the Fe(II) form is oxidized in the presence of product. Together, these results suggest that product exchange occurs only when the mononuclear iron is reduced. Stopped-flow and rapid scan analyses monitoring the oxidation of the Rieske cluster during the single turnover reaction show that it occurs in three phases that are kinetically competent for catalysis. The rate of each phase was found to be dependent on the type of substrate present, suggesting that the substrate influences the rate of electron transfer between the metal clusters. The participation of substrate in the oxygen activation reaction suggests a new aspect of the mechanism of this process by the Rieske dioxygenase class.

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.

FTIR studies of benzoate adsorption on the Au(111) electrode

Abstract Subtractively normalized interfacial Fourier transform infrared spectroscopy (SNIFTIRS) has been used to examine (i) the orientation and coordination of benzoate on the Au(111) electrode; (ii) the dependence of the IR band intensity on the surface coverage; and (iii) the chemical stability of benzoate at the Au(111) surface. The results indicate that orientation of adsorbed benzoate changes from a nearly flat co-ordination at a negatively charged surface to a tilted position at positive charge densities. The transition in the orientation of the adsorbed molecule occurs in the vicinity of the potential of zero charge in the solution investigated. The integrated IR band intensities correlate very well with the surface concentration of benzoate obtained from chronocoulometric measurements. An overall agreement between the spectroscopic and electrochemical results is excellent. The spectroscopic data demonstrate that no decomposition or oxidation of benzoate occurred at the Au(111) electrode surface over the potential range investigated.

Energetics of product formation during anaerobic degradation of phthalate isomers and benzoate

Methanogenic enrichment cultures grown on phthalate, isophthalate and terephthalate were incubated with the corresponding phthalate isomer on which they were grown, and a mixture of benzoate and the phthalate isomer. All cultures were incubated with bromoethanosulfonate to inactivate the methanogens in the mixed culture. Thus, product formation during fermentation of the aromatic substrates could be studied. It was found that reduction equivalents generated during oxidation of the aromatic substrates to acetate were incorporated in benzoate under formation of carboxycyclohexane. During fermentation of the phthalate isomers, small amounts of benzoate were detected, suggesting that the initial step in the anaerobic degradation of the phthalate isomers is decarboxylation to benzoate. Gibbs free energy analyses indicated that during degradation of the phthalate isomers, benzoate, carboxycyclohexane, acetate and molecular hydrogen accumulated in such amounts that both the reduction and oxidation of benzoate yielded a constant and comparable amount of energy of approximately 30 kJ mol−1. Based on these observations it is suggested that within narrow energetic limits, oxidation and reduction of benzoate may proceed simultaneously. Whether this is controlled by the Gibbs free energy change for carboxycyclohexane oxidation remains unclear.

Energetics of product formation during anaerobic degradation of phthalate isomers and benzoate

Methanogenic enrichment cultures grown on phthalate, isophthalate and terephthalate were incubated with the corresponding phthalate isomer on which they were grown, and a mixture of benzoate and the phthalate isomer. All cultures were incubated with bromoethanosulfonate to inactivate the methanogens in the mixed culture. Thus, product formation during fermentation of the aromatic substrates could be studied. It was found that reduction equivalents generated during oxidation of the aromatic substrates to acetate were incorporated in benzoate under formation of carboxycyclohexane. During fermentation of the phthalate isomers, small amounts of benzoate were detected, suggesting that the initial step in the anaerobic degradation of the phthalate isomers is decarboxylation to benzoate. Gibbs free energy analyses indicated that during degradation of the phthalate isomers, benzoate, carboxycyclohexane, acetate and molecular hydrogen accumulated in such amounts that both the reduction and oxidation of benzoate yielded a constant and comparable amount of energy of approximately 30 kJ mol −1. Based on these observations it is suggested that within narrow energetic limits, oxidation and reduction of benzoate may proceed simultaneously. Whether this is controlled by the Gibbs free energy change for carboxycyclohexane oxidation remains unclear.

Reductive dehalogenation and mineralization of 3-chlorobenzoate in the presence of sulfate by microorganisms from a methanogenic aquifer

We investigated the anaerobic biodegradation of 3-chlorobenzoate (3CBz) by microorganisms from an aquifer where chloroaromatic compounds were previously found to resist decay in the presence of sulfate. After a lengthy lag period, 3CBz was degraded in the presence of sulfate and concurrently with sulfate reduction. Chlorine removal from 2,5- or 3,5-dichlorobenzoates and the transient appearance of benzoate from 3CBz confirmed that reductive dehalogenation was the initial fate process for these substrates. Sulfate did not influence 3CBz degradation rates in acclimated enrichment cultures but accelerated the development of 3CBz degradation activity in fresh transfers. Benzoate degradation was more rapid in the presence of sulfate regardless of the enrichment history. Nitrate, sulfite, and a headspace of air inhibited 3CBz dehalogenation, while thiosulfate had no effect. Mass balance determinations revealed that 71 to 107% of the theoretically expected amount of methane was produced from 3CBz and benzoate oxidation in the absence of sulfate. In parallel cultures containing 15 mM sulfate, methanogenesis was reduced to 48 to 71% of that theoretically expected, while sulfate reduction accounted for 12 to 50% of the reducing equivalents. In either the presence or absence of sulfate, steady-state dissolved hydrogen concentrations were similar to those reported for sulfate-reducing or methanogenic environments, respectively. Molybdate inhibited sulfate reduction and 3CBz dehalogenation to a similar extent but did not affect benzoate biodegradation. Sulfate-dependent 3CBz biodegradation was not observed. We conclude that reductive dehalogenation and sulfate reduction occur concurrently in these enrichments and that the sulfate-dependent stimulation in fresh transfers was likely due to the acceleration of benzoate oxidation.

Simultaneous Determination of Chromate and Aromatic Hydrocarbons in Environmental Samples by Capillary Electrophoresis

An analytical method was developed to determine simultaneously, the inorganic anion CrO2− 4, and organic aromatic compounds including benzoate, 2-Cl-benzoate, phenol, m-cresol and o-/p-cresol by capillary electrophoresis (CE). Chromate and the aromatics were separated in a relatively short time with the use of a tetradecyltrimethylammonium bromide (TTAB) modified buffer solution. The detection limits of all the analytes were in the sub mg/L level with the consumption of a very small volume of sample (< 0.06 μL). Calibration curves with high regression coefficient (r2) values (0.999–1.000) were obtained within two orders of magnitude of concentrations. Factors affecting the separation and the determination of chromate and the organic aromatics, such as the buffer electrolyte concentration, the voltage applied and the TTAB concentration, were investigated. The effect of TTAB on the direction and the magnitude of the electroosmotic flow (EOF) was also discussed. A study of the microbial catalyzed chromate reduction coupled with benzoate oxidation demonstrated the application potential of the proposed method.

Acute Toxicity of Cresols to Pseudomonas — An Initial Oxygen Uptake Method

AbstractAcute toxicity of cresols to both Pseudomonas I and II was estimated by an initial oxygen uptake method. Inhibition studies of toluene and cresols on the oxidation of either benzoate by Pseudomnas I or phenol by Pseudomonas II were analyzed and expressed as oxygen uptake rates. Double reciprocal plots for the inhibiton by cresols of oxygen uptake in Pseudomonas, two physical constants, Vmaxi and Ki, were obtained. The Vmaxi of o−, m− and p‐cresol were 80%, 81% and 57% of Vmax in Pseudomnas I, and 10%, 25% and 36% in Pseudomonas II, respectively. Thus, the toxicity to Pseudomonas I decreases in the order p‐ &gt; o‐ ≥ m‐cresol, whereas to Pseudomonas II, the order is changed to o‐ &gt; m‐ &gt; p‐cresol. This difference in the toxicity order is probably due to the allosteric effect of p‐cresol towards Pseudomonas II.Inasmuch as most compounds inhibit noncompetively, the relative toxicity of different compounds can be estimated by a new toxicity parameter RI (relative inhibition) which is defined as 100/Ki. By comparing the RI value of each compound, the toxicity to Pseudomonas I decreases in the order m‐chlorophenol &gt; p‐cresol &gt; p‐chlorophenol &gt; o‐cresol ≥ m‐cresol &gt; o‐chlorophenol &gt; toluene &gt; phenol.

Microbial Reduction of Cr(VI) during Anaerobic Degradation of Benzoate

A series of microcosms and enrichments were conducted to evaluate the potential for microbially mediated Cr(VI) reduction linked to benzoate catabolism. Bacterial degradation of benzoate with the transport of electrons to Cr(VI) was achieved using nitrate or molecular oxygen as an initial stimulator. After depletion of nitrate or oxygen, microcosms and enrichments still retained the capacity for benzoate degradation linked to Cr(VI) reduction. Experiments demonstrated that benzoate degradation occurred concurrently with the reduction of Cr(VI) and was strongly dependent on the presence of Cr(VI). Benzoate degradation proceeded as long as Cr(VI) was present, ceased when Cr(VI) was completely removed, and continued once Cr(VI) was added. Furthermore, the observed benzoate removal, linearly correlated with Cr(VI) consumed, and the stoichiometric ratio were in good agreement to the theoretical ratio for the complete oxidation of benzoate to carbon dioxide coupled to the reduction of Cr(VI) to Cr(III). The addition of nitrate up to a concentration of 5.0 mM did not inhibit Cr(VI) reduction but enhanced benzoate degradation in the enrichments. This process may provide the opportunity for in-situ biotreatment of Cr(VI) in subsurface environments.

Degradation of BTEX and their aerobic metabolites by indigenous microorganisms under nitrate reducing conditions

Batch incubations, seeded with four different aquifer materials, were used to survey the catabolic capacity of indigenous microorganisms under nitrate reducing conditions. Benzene, toluene, ethylbenzene, xylenes (BTEX), and selected potential metabolites of their incomplete aerobic degradation, were tested as substrates for nitrate-based respiration. Toluene and its potential aerobic metabolites, benzoate, protocatechuate, 3-methylcatechol, 4-methylcatechol, succinate, and adipate were degraded in strictly anoxic (O2 < 0.1 mg/l) nitrate reducing incubations. Toluene degradation was directly coupled to nitrate reduction. Ortho-xylene removal was toluene dependent. Meta- and para-xylenes were degraded in nitrate reducing enrichments from only one of the four aquifer samples. Benzene, ethylbenzene, catechol and gentisate were not degraded within up to four months in any of the incubations, even though nitrate reduction occurred. Anaerobic benzene degradation was not observed. Incubations receiving nitrate as an adjunct electron acceptor to oxygen degraded significantly more benzene than incubations amended with only oxygen, although benzene was only degraded until the dissolved oxygen was depleted. Possibly, more oxygen was available to degrade benzene when nitrate was added because denitrifiers utilizing nitrate as terminal electron acceptor oxidized benzoate, which had been added to increase the biochemical oxygen demand of the system. Benzoate oxidation with nitrate apparently spared oxygen for benzene degradation.

Degradation of BTEX and their aerobic metabolites by indigenous microorganisms under nitrate reducing conditions

Batch incubations, seeded with four different aquifer materials, were used to survey the catabolic capacity of indigenous microorganisms under nitrate reducing conditions. Benzene, toluene, ethylbenzene, xylenes (BTEX), and selected potential metabolites of their incomplete aerobic degradation, were tested as substrates for nitrate-based respiration. Toluene and its potential aerobic metabolites, benzoate, protocatechuate, 3-methylcatechol, 4-methylcatechol, succinate, and adipate were degraded in strictly anoxic (O2 &amp;lt; 0.1 mg/l) nitrate reducing incubations. Toluene degradation was directly coupled to nitrate reduction. Ortho-xylene removal was toluene dependent. Meta- and para-xylenes were degraded in nitrate reducing enrichments from only one of the four aquifer samples. Benzene, ethylbenzene, catechol and gentisate were not degraded within up to four months in any of the incubations, even though nitrate reduction occurred. Anaerobic benzene degradation was not observed. Incubations receiving nitrate as an adjunct electron acceptor to oxygen degraded significantly more benzene than incubations amended with only oxygen, although benzene was only degraded until the dissolved oxygen was depleted. Possibly, more oxygen was available to degrade benzene when nitrate was added because denitrifiers utilizing nitrate as terminal electron acceptor oxidized benzoate, which had been added to increase the biochemical oxygen demand of the system. Benzoate oxidation with nitrate apparently spared oxygen for benzene degradation.

Benzoyl-coenzyme-A 3-monooxygenase, a flavin-dependent hydroxylase. Purification, some properties and its role in aerobic benzoate oxidation via gentisate in a denitrifying bacterium.

A new variant of aerobic benzoate degradation has been found in a denitrifying bacterium in which benzoyl-CoA is the first intermediate [Altenschmidt, U., Oswald, B., Steiner, E., Herrmann, H. & Fuchs, G. (1993) New aerobic benzoate oxidation pathway via benzoyl-coenzyme A and 3-hydroxybenzoyl-coenzyme A in a denitrifying Pseudomonas sp, J. Bacteriol. 175, 4851-4858)]. The initial reaction is catalyzed by benzoate-CoA ligase (AMP-forming), converting benzoate into benzoyl-CoA. The next step is 3-hydroxylation of benzoyl-CoA to 3-hydroxybenzoyl-CoA catalyzed by a flavin-nucleotide-dependent monooxygenase, benzoyl-CoA 3-monooxygenase. This novel enzyme has been purified and studied. It is specific for NADPH and requires the presence of a flavin nucleotide for activity; both FAD or FMN function similarly well as cofactor. Only benzoyl-CoA, but not benzoate, is hydroxylated. The protein is a monomer of M(r) 65,000 and is induced when cells are grown aerobically with benzoate. 3-Hydroxybenzoyl-CoA is further hydroxylated para to the hydroxyl group affording 2,5-dihydroxybenzoate (gentisate). This reaction requires another monooxygenase, 3-hydroxybenzoyl-CoA 6-monooxygenase, which is unspecific specific with respect to the pyridine nucleotide. Cells contain a second 6-monooxygenase activity which acts on free 3-hydroxybenzoate. Based on these and other data, the outlines of the new aerobic benzoate pathway have been deduced. The proposed intermediates are benzoyl-CoA, 3-hydroxybenzoyl-CoA, gentisate, maleylpyruvate, fumarylpyruvate and fumarate plus pyruvate.

Evidence of reversed electron transport in syntrophic butyrate or benzoate oxidation by Syntrophomonas wolfei and Syntrophus buswellii

Syntrophomonas wolfei and Syntrophus buswellii were grown with butyrate or benzoate in a defined binary coculture with Methanospirillum hungatei. Both strains also grew independent of the partner bacteria with crotonate as substrate. Localization of enzymes involved in butyrate oxidation by S. wolfei revealed that ATP synthase, hydrogenase, and butyryl-CoA dehydrogenase were at least partially membrane-associated whereas 3-hydroxybutyryl-CoA dehydrogenase and crotonase were entirely cytoplasmic. Inhibition experiments with copper chloride indicated that hydrogenase faced the outer surface of the cytoplasmic membrane. Suspensions of butyrate-or benzoate-grown cells of either strain accumulated hydrogen during oxidation of butyrate or benzoate to a low concentration that was thermodynamically in equilibrium with calculated reaction energetics. The protonophore carbonylcyanide m-chlorophenyl-hydrazone (CCCP) and the proton-translocating ATPase inhibitor N,N′dicyclohexylcarbodiimide (DCCD) both specifically inhibited hydrogen formation from butyrate or benzoate at low concentrations, whereas hydrogen formation from crotonate was not affected. A menaquinone was extracted from cells of S. wolfei and S. buswellii grown syntrophically in a binary methanogenic culture. The results indicate that a proton-potential-driven process is involved in hydrogen release from butyrate or benzoate oxidation.

Evidence of reversed electron transport in syntrophic butyrate or benzoate oxidation by Syntrophomonas wolfei and Syntrophus buswellii

Syntrophomonas wolfei and Syntrophus buswellii were grown with butyrate or benzoate in a defined binary coculture with Methanospirillum hungatei. Both strains also grew independent of the partner bacteria with crotonate as substrate. Localization of enzymes involved in butyrate oxidation by S. wolfei revealed that ATP synthase, hydrogenase, and butyryl-CoA dehydrogenase were at least partially membrane-associated whereas 3-hydroxybutyryl-CoA dehydrogenase and crotonase were entirely cytoplasmic. Inhibition experiments with copper chloride indicated that hydrogenase faced the outer surface of the cytoplasmic membrane. Suspensions of butyrate-or benzoate-grown cells of either strain accumulated hydrogen during oxidation of butyrate or benzoate to a low concentration that was thermodynamically in equilibrium with calculated reaction energetics. The protonophore carbonylcyanide m-chlorophenyl-hydrazone (CCCP) and the proton-translocating ATPase inhibitor N,N′dicyclohexylcarbodiimide (DCCD) both specifically inhibited hydrogen formation from butyrate or benzoate at low concentrations, whereas hydrogen formation from crotonate was not affected. A menaquinone was extracted from cells of S. wolfei and S. buswellii grown syntrophically in a binary methanogenic culture. The results indicate that a proton-potential-driven process is involved in hydrogen release from butyrate or benzoate oxidation.

In vivo reactivation of catechol 2,3-dioxygenase mediated by a chloroplast-type ferredoxin: a bacterial strategy to expand the substrate specificity of aromatic degradative pathways.

The meta-cleavage operon of the TOL plasmid pWW0 of Pseudomonas putida contains 13 genes responsible for the oxidation of benzoate and toluates to Krebs cycle intermediates via estradiol (meta) cleavage of (methyl)catechol. The functions of all the genes are known with the exception of xylT. We constructed pWW0 mutants defective in the xylT gene, and found that these mutants were not able to grow on p-toluate while they were still capable of growing on benzoate and m-toluate. In the xylT mutants, all the meta-cleavage enzymes were induced by p-toluate with the exception of catechol 2,3-dioxygenase whose activity was 1% of the p-toluate-induced activity in wild-type cells. Addition of 4-methylcatechol to m-toluate-grown wild-type and xylT cells resulted in the inactivation of catechol 2,3-dioxygenase in these cells. In the wild-type strain but not in the xylT mutant, the catechol 2,3-dioxygenase activity was regenerated in a short time. The regeneration of the catechol 2,3-dioxygenase activity was also observed in H2O2-treated wild-type cells, but not in H2O2-treated xylT cells. We concluded that the xylT product is required for the regeneration of catechol 2,3-dioxygenase.