Anaerobic metabolism of 3-hydroxybenzoate by the denitrifying bacterium Thauera aromatica

The anaerobic metabolism of 3-hydroxybenzoate was studied in the denitrifying bacterium Thauera aromatica. Cells grown with this substrate were adapted to grow with benzoate but not with 4-hydroxybenzoate. Vice versa, 4-hydroxybenzoate-grown cells did not utilize 3-hydroxybenzoate. The first step in 3-hydroxybenzoate metabolism is a coenzyme A (CoA) thioester formation, which is catalyzed by an inducible 3-hydroxybenzoate-CoA ligase. The enzyme was purified and characterized. Further metabolism of 3-hydroxybenzoyl-CoA by cell extract required MgATP and was coupled to the oxidation of 2 mol of reduced viologen dyes per mol of substrate added. Purification of the 3-hydroxybenzoyl-CoA reducing enzyme revealed that this activity was due to benzoyl-CoA reductase, which reduced the 3-hydroxy analogue almost as efficiently as benzoyl-CoA. The further metabolism of the alicyclic dienoyl-CoA product containing the hydroxyl substitution obviously required additional specific enzymes. Comparison of the protein pattern of 3-hydroxybenzoate-grown cells with benzoate-grown cells revealed several 3-hydroxybenzoate-induced proteins; the N-terminal amino acid sequences of four induced proteins were determined and the corresponding genes were identified and sequenced. A cluster of six adjacent genes contained the genes for substrate-induced proteins 1 to 3; this cluster may not yet be complete. Protein 1 is a short-chain alcohol dehydrogenase. Protein 2 is a member of enoyl-CoA hydratase enzymes. Protein 3 was identified as 3-hydroxybenzoate-CoA ligase. Protein 4 is another member of the enoyl-CoA hydratases. In addition, three genes coding for enzymes of beta-oxidation were present. The anaerobic 3-hydroxybenzoate metabolism here obviously combines an enzyme (benzoyl-CoA reductase) and electron carrier (ferredoxin) of the general benzoyl-CoA pathway with enzymes specific for the 3-hydroxybenzoate pathway. This raises some questions concerning the regulation of both pathways.     

Genetic diversity of benzoyl coenzyme A reductase genes detected in denitrifying isolates and estuarine sediment communities

Benzoyl coenzyme A (benzoyl-CoA) reductase is a central enzyme in the anaerobic degradation of organic carbon, which utilizes a common intermediate (benzoyl-CoA) in the metabolism of many aromatic compounds. The diversity of benzoyl-CoA reductase genes in denitrifying bacterial isolates capable of degrading aromatic compounds and in river and estuarine sediment samples from the Arthur Kill in New Jersey and the Chesapeake Bay in Maryland was investigated. Degenerate primers were developed from the known benzoyl-CoA reductase genes from Thauera aromatica, Rhodopseudomonas palustris, and Azoarcus evansii. PCR amplification detected benzoyl-CoA reductase genes in the denitrifying isolates belonging to alpha-, beta-, or gamma-Proteobacteria as well as in the sediment samples. Phylogenetic analysis, sequence similarity comparison, and conserved indel determination grouped the new sequences into either the bcr type (found in T. aromatica and R. palustris) or the bzd type (found in A. evansii). All the Thauera strains and the isolates from the genera Acidovorax, Bradyrhizobium, Paracoccus, Ensifer, and Pseudomonas had bcr-type benzoyl-CoA reductases with amino acid sequence similarities of more than 97%. The genes detected from Azarocus strains were assigned to the bzd type. A total of 50 environmental clones were detected from denitrifying consortium and sediment samples, and 28 clones were assigned to either the bcr or the bzd type of benzoyl-CoA reductase genes. Thus, we could determine the genetic capabilities for anaerobic degradation of aromatic compounds in sediment communities of the Chesapeake Bay and the Arthur Kill on the basis of the detection of two types of benzoyl-CoA reductase genes. The detected genes have future applications as genetic markers to monitor aromatic compound degradation in natural and engineered ecosystems.     

Two distinct pathways for anaerobic degradation of aromatic compounds in the denitrifying bacterium Thauera aromatica strain AR-1

Denitrifying bacteria degrade many different aromatic compounds anaerobically via the well-described benzoyl-CoA pathway. We have shown recently that the denitrifiers Azoarcus anaerobius and Thauera aromatica strain AR-1 use a different pathway for anaerobic degradation of resorcinol (1,3-dihydroxybenzene) and 3,5-dihydroxybenzoate, respectively. Both substrates are converted to hydroxyhydroquinone (1,2,4-trihydroxybenzene). In the membrane fraction of T. aromatica strain AR-1 cells grown with 3,5-dihydroxybenzoate, a hydroxyhydroquinone-dehydrogenating activity of 74 nmol min(-1)(mg protein)-1 was found. This activity was significantly lower in benzoate-grown cells. Benzoate-grown cells were not induced for degradation of 3,5-dihydroxybenzoate, and cells grown with 3,5-dihydroxybenzoate degraded benzoate only at a very low rate. With a substrate mixture of benzoate plus 3,5-dihydroxybenzoate, the cells showed diauxic growth. Benzoate was degraded first, while complete degradation of 3,5-dihydroxybenzoate occurred only after a long lag phase. The 3,5-dihydroxybenzoate-oxidizing and the hydroxyhydroquinone-dehydrogenating activities were fully induced only during 3,5-dihydroxybenzoate degradation. Synthesis of benzoyl-CoA reductase appeared to be significantly lower in 3,5-dihydroxybenzoate-grown cells as shown by immunoblotting. These results confirm that T. aromatica strain AR-1 harbors, in addition to the benzoyl-CoA pathway, a second, mechanistically distinct pathway for anaerobic degradation of aromatic compounds. This pathway is inducible and subject to catabolite repression by benzoate.     

Cyclohexa-1,5-diene-1-carbonyl-coenzyme A (CoA) hydratases of Geobacter metallireducens and Syntrophus aciditrophicus: Evidence for a common benzoyl-CoA degradation pathway in facultative and strict anaerobes

In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyclohexa-1,5-diene-1-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of beta-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophicus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA (Km, 80 and 35 microM; Vmax, 350 and 550 micromol min(-1) mg(-1), respectively). Neither enzyme had significant activity with cyclohex-1-ene-1-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes.     

Anaerobic degradation of 2-aminobenzoic acid (anthranilic acid) via benzoyl-coenzyme A (CoA) and cyclohex-1-enecarboxyl-CoA in a denitrifying bacterium.

The enzymes catalyzing the initial reactions in the anaerobic degradation of 2-aminobenzoic acid (anthranilic acid) were studied with a denitrifying Pseudomonas sp. anaerobically grown with 2-aminobenzoate and nitrate as the sole carbon and energy sources. Cells grown on 2-aminobenzoate are simultaneously adapted to growth with benzoate, whereas cells grown on benzoate degrade 2-aminobenzoate several times less efficiently than benzoate. Evidence for a new reductive pathway of aromatic metabolism and for four enzymes catalyzing the initial steps is presented. The organism contains 2-aminobenzoate-coenzyme A ligase (2-aminobenzoate-CoA ligase), which forms 2-aminobenzoyl-CoA. 2-Aminobenzoyl-CoA is then reductively deaminated to benzoyl-CoA by an oxygen-sensitive enzyme, 2-aminobenzoyl-CoA reductase (deaminating), which requires a low potential reductant [Ti(III)]. The specific activity is 15 nmol of 2-aminobenzoyl-CoA reduced min-1 mg-1 of protein at an optimal pH of 7. The two enzymes are induced by the substrate under anaerobic conditions only. Benzoyl-CoA is further converted in vitro by reduction with Ti(III) to six products; the same products are formed when benzoyl-CoA or 2-aminobenzoyl-CoA is incubated under reducing conditions. Two of them were identified preliminarily. One product is cyclohex-1-enecarboxyl-CoA, the other is trans-2-hydroxycyclohexane-carboxyl-CoA. The complex transformation of benzoyl-CoA is ascribed to at least two enzymes, benzoyl-CoA reductase (aromatic ring reducing) and cyclohex-1-enecarboxyl-CoA hydratase. The reduction of benzoyl-CoA to alicyclic compounds is catalyzed by extracts from cells grown anaerobically on either 2-aminobenzoate or benzoate at almost the same rate (10 to 15 nmol min-1 mg-1 of protein). In contrast, extracts from cells grown anaerobically on acetate or grown aerobically on benzoate or 2-aminobenzoate are inactive. This suggests a sequential induction of the enzymes.     

Genetic Diversity of Benzoyl Coenzyme A Reductase Genes Detected in Denitrifying Isolates and Estuarine Sediment Communities

Benzoyl coenzyme A (benzoyl-CoA) reductase is a central enzyme in the anaerobic degradation of organic carbon, which utilizes a common intermediate (benzoyl-CoA) in the metabolism of many aromatic compounds. The diversity of benzoyl-CoA reductase genes in denitrifying bacterial isolates capable of degrading aromatic compounds and in river and estuarine sediment samples from the Arthur Kill in New Jersey and the Chesapeake Bay in Maryland was investigated. Degenerate primers were developed from the known benzoyl-CoA reductase genes from Thauera aromatica, Rhodopseudomonas palustris, and Azoarcus evansii. PCR amplification detected benzoyl-CoA reductase genes in the denitrifying isolates belonging to α-, β-, or γ-Proteobacteria as well as in the sediment samples. Phylogenetic analysis, sequence similarity comparison, and conserved indel determination grouped the new sequences into either the bcr type (found in T. aromatica and R. palustris) or the bzd type (found in A. evansii). All the Thauera strains and the isolates from the genera Acidovorax, Bradyrhizobium, Paracoccus, Ensifer, and Pseudomonas had bcr-type benzoyl-CoA reductases with amino acid sequence similarities of more than 97%. The genes detected from Azarocus strains were assigned to the bzd type. A total of 50 environmental clones were detected from denitrifying consortium and sediment samples, and 28 clones were assigned to either the bcr or the bzd type of benzoyl-CoA reductase genes. Thus, we could determine the genetic capabilities for anaerobic degradation of aromatic compounds in sediment communities of the Chesapeake Bay and the Arthur Kill on the basis of the detection of two types of benzoyl-CoA reductase genes. The detected genes have future applications as genetic markers to monitor aromatic compound degradation in natural and engineered ecosystems.     

Properties of 2-oxoglutarate:ferredoxin oxidoreductase from Thauera aromatica and its role in enzymatic reduction of the aromatic ring

Benzoyl coenzyme A (benzoyl-CoA) reductase is a key enzyme in the anaerobic metabolism of aromatic compounds catalyzing the ATP-driven reductive dearomatization of benzoyl-CoA. The enzyme from Thauera aromatica uses a reduced 2[4Fe-4S] ferredoxin as electron donor. In this work, we identified 2-oxoglutarate:ferredoxin oxidoreductase (KGOR) as the ferredoxin reducing enzyme. KGOR activity was increased 10- to 50-fold in T. aromatica cells grown under denitrifying conditions on an aromatic substrate compared to that of cells grown on nonaromatic substrates. The enzyme was purified from soluble extracts by a 60-fold enrichment with a specific activity of 4.8 micromol min(-1) mg(-1). The native enzyme had a molecular mass of 200 +/- 20 kDa (mean +/- standard deviation) and consisted of two subunits with molecular masses of 66 and 34 kDa, suggesting an (alphabeta)(2) composition. The UV/visible spectrum was characteristic for an iron-sulfur protein; the enzyme contained 8.3 +/- 0.5 mol of Fe, 7.2 +/- 0.5 mol of acid-labile sulfur, and 1.6 +/- 0.2 mol of thiamine diphosphate (TPP) per mol of protein. The high specificity for 2-oxoglutarate and the low K(m) for ferredoxin ( approximately 10 microM) indicated that both are the in vivo substrates of the enzyme. KGOR catalyzed the isotope exchange between (14)CO(2) and C(1) of 2-oxoglutarate, representing a typical reversible partial reaction of 2-oxoacid oxidoreductases. The two genes coding for the two subunits of KGOR were found adjacent to the gene cluster coding for enzymes and ferredoxin of the catabolic benzoyl-CoA pathway. Sequence comparisons with other 2-oxoacid oxidoreductases indicated that KGOR from T. aromatica belongs to the Halobacterium type of 2-oxoacid oxidoreductases, which lack a ferredoxin-like module which contains two additional [4Fe-4S](1+/2+) clusters/monomer. Using purified KGOR, ferredoxin, and benzoyl-CoA reductase, the 2-oxoglutarate-driven reduction of benzoyl-CoA was shown in vitro. This demonstrates that ferredoxin acts as an electron shuttle between the citric acid cycle and benzoyl-CoA reductase by coupling the oxidation of the end product of the benzoyl-CoA pathway, acetyl-CoA, to the reduction of the aromatic ring.     

Differential induction of enzymes involved in anaerobic metabolism of aromatic compounds in the denitrifying bacterium Thauera aromatica

Differential induction of enzymes involved in anaerobic metabolism of aromatic substrates was studied in the denitrifying bacterium Thauera aromatica. This metabolism is divided into (1) peripheral reactions transforming the aromatic growth substrates to the common intermediate benzoyl-CoA, (2) the central benzoyl-CoA pathway comprising ring-reduction of benzoyl-CoA and subsequent β-oxidation to 3-hydroxypimelyl-CoA, and (3) the pathway of β-oxidation of 3-hydroxypimelyl-CoA to three acetyl-CoA and CO₂. Regulation was studied by three methods. 1. Determination of protein patterns of cells grown on different substrates. This revealed several strongly substrate-induced polypeptides that were missing in cells grown on benzoate or other intermediates of the respective metabolic pathways. 2. Measurement of activities of known enzymes involved in this metabolism in cells grown on different substrates. The enzyme pattern found is consistent with the regulatory pattern deduced from simultaneous adaptation of cells to utilisation of other aromatic substrates. 3. Immunological detection of catabolic enzymes in cells grown on different substrates. Benzoate-CoA ligase and 4-hydroxybenzoate-CoA ligase were detected only in cells yielding the respective enzyme activity. However, presence of the subunits of benzoyl-CoA reductase and 4-hydroxybenzoyl-CoA reductase was also recorded in some cell batches lacking enzyme activity. This possibly indicates an additional level of regulation on protein level for these two reductases. Received: 22 December 1997 / Accepted: 12 May 1998     

Anaerobic degradation of toluene in denitrifying Pseudomonas sp.: indication for toluene methylhydroxylation and benzoyl-CoA as central aromatic intermediate

The anaerobic degradation of toluene has been studied with whole cells and by measuring enzyme activities. Cultures of Pseudomonas strain K 172 were grown in mineral medium up to a cell density of 0.5 g of dry cells per liter in fed-batch culture with toluene and nitrate as the sole carbon and energy sources. A molar growth yield of 57 g of cell dry matter formed per mol toluene totally consumed was determined. The mean generation time was 24 h. The redox balance between toluene consumed (oxidation and cell material synthesis) and nitrate consumed (reduction to nitrogen gas and assimilation as NH₃) was 77% of expectation if toluene was completely oxidized; this indicated that the major amount of toluene was mineralized to CO₂. It was tested whether the initial reaction in anaerobic toluene degradation was a carboxylation or a dehydrogenation (anaerobic hydroxylation); the hypothetical carboxylated or hydroxylated intermediates were tested with whole cells applying the method of simultanous adaptation: cells pregrown on toluene degraded benzyl alcohol, benzaldehyde, and benzoic acid without lag, 4-hydroxybenzoate and p-cresol with a 90 min lag phase and phenylacetate after a 200 min lag phase. The cells were not at all adapted to degrade 2-methylbenzoate, 4-methylbenzoate, o-cresol, and m-cresol, nor did these compounds support growth within a few days after inoculation with cells grown on toluene. In extracts of cells anaerobically grown on toluene, benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase, and benzoyl-CoA synthetase (AMP forming) activities were present. The data (1) conclusively show anaerobic growth of a pure culture on tolucne; (2) suggest that toluene is anaerobically degraded via benzoyl-CoA; (3) imply that water functions as the source of the hydroxyl group in a toluene methylhydroxylase reaction.     

Initial Reactions in Anaerobic Oxidation of m-Xylene by the Denitrifying Bacterium Azoarcus sp. Strain T

The initial enzymatic steps in anaerobic m-xylene oxidation were studied in Azoarcus sp. strain T, a denitrifying bacterium capable of mineralizing m-xylene via 3-methylbenzoate. Permeabilized cells of m-xylene-grown Azoarcus sp. strain T catalyzed the addition of m-xylene to fumarate to form (3-methylbenzyl)succinate. In the presence of succinyl coenzyme A (CoA) and nitrate, (3-methylbenzyl)succinate was oxidized to E-(3-methylphenyl)itaconate (or a closely related isomer) and 3-methylbenzoate. Kinetic studies conducted with permeabilized cells and whole-cell suspensions of m-xylene-grown Azoarcus sp. strain T demonstrated that the specific rate of in vitro (3-methylbenzyl)succinate formation accounts for at least 15% of the specific rate of in vivo m-xylene consumption. Based on these findings, we propose that Azoarcus sp. strain T anaerobically oxidizes m-xylene to 3-methylbenzoate (or its CoA thioester) via (3-methylbenzyl)succinate and E-(3-methylphenyl)itaconate (or its CoA thioester) in a series of reactions that are analogous to those recently proposed for anaerobic toluene oxidation to benzoyl-CoA. A deuterium kinetic isotope effect was observed in the (3-methylbenzyl)succinate synthase reaction (and the benzylsuccinate synthase reaction), suggesting that a rate-determining step in this novel fumarate addition reaction involves breaking a C-H bond.     

Anaerobic metabolism of l-phenylalanine via benzoyl-CoA in the denitrifying bacterium Thauera aromatica

The anaerobic metabolism of phenylalanine was studied in the denitrifying bacterium Thauera aromatica, a member of the β-subclass of the Proteobacteria. Phenylalanine was completely oxidized and served as the sole source of cell carbon. Evidence is presented that degradation proceeds via benzoyl-CoA as the central aromatic intermediate; the aromatic ring-reducing enzyme benzoyl-CoA reductase was present in cells grown on phenylalanine. Intermediates in phenylalanine oxidation to benzoyl-CoA were phenylpyruvate, phenylacetaldehyde, phenylacetate, phenylacetyl-CoA, and phenylglyoxylate. The required enzymes were detected in extracts of cells grown with phenylalanine and nitrate. Oxidation of phenylalanine to benzoyl-CoA was catalyzed by phenylalanine transaminase, phenylpyruvate decarboxylase, phenylacetaldehyde dehydrogenase (NAD⁺), phenylacetate-CoA ligase (AMP-forming), enzyme(s) oxidizing phenylacetyl-CoA to phenylglyoxylate with nitrate, and phenylglyoxylate:acceptor oxidoreductase. The capacity for phenylalanine oxidation to phenylacetate was induced during growth with phenylalanine. Evidence is provided that α-oxidation of phenylacetyl-CoA is catalyzed by a membrane-bound enzyme. This is the first report on the complete anaerobic degradation of an aromatic amino acid and the regulation of this process. Received: 6 March 1997 / Accepted: 16 May 1997     

A catabolic gene cluster for anaerobic benzoate degradation in methanotrophic microbial Black Sea mats

A microbial mat from the Black Sea shelf was analyzed by a metagenomic approach. While the habitat and its microbial community are characterized by anaerobic methane oxidation, a 79 kb contiguous DNA sequence obtained from the same mat provided first evidence for the concomitant presence of the capacity for anaerobic benzoate degradation. Benzoyl-CoA is one central intermediate of anaerobic aromatic degradation, among others. Within a stretch of 31 kb, all genes required for the complete pathway of anaerobic benzoate degradation (catabolic island) were identified, including the four subunits of the key enzyme benzoyl-CoA reductase (bcrCBAD), which catalyzes the ATP-driven 2-electron reduction of the aromatic ring. Genes for a ketoacid:acceptor oxidoreductase (korABC) and a ferredoxin (fdx), which are required for generation of a suitable electron donor, were also detected. The majority of the identified catabolic gene products are most similar to their respective orthologs from the denitrifying freshwater bacterium Azoarcus evansii, and the genes are also similarly organized. Due to the lack of established markers, the phylogenetic affiliation of the source organism remains unclear. The presented findings indicate that the metabolic diversity of the Black Sea mat is wider than currently known and that probably other bacteria than those of the methane-oxidizing consortia contribute to aromatic degradation in this anoxic habitat.     

Anaerobic metabolism of 2-hydroxybenzoic acid (salicylic acid) by a denitrifying bacterium

The anaerobic metabolism of 2-hydroxybenzoic acid (salicylic acid) was studied in a denitrifying bacterium. Cells grown with 2-hydroxybenzoate were simultaneously adapted to degrade benzoate. Extract of these cells formed benzoate or benzoyl-CoA when incubated under reducing conditions with salicylate, MgATP, and coenzyme A, suggesting a degradation of 2-hydroxybenzoate via benzoate or benzoyl-CoA. This suggestion was supported by enzyme activity measurements. In extracts of 2-hydroxybenzoate-grown cells, the following enzyme activities were detected: two CoA ligases, one specific for 2-hydroxybenzoate, the other for benzoate, and two different enzyme activities catalyzing the reductive transformation of 2-hydroxybenzoyl-CoA. These findings suggest a degradation of salicylic acid by two new enzymes, 2-hydroxybenzoate-CoA ligase (AMP-forming) and 2-hydroxybenzoyl-CoA reductase (dehydroxylating), catalyzing (1) 2-hydroxybenzoate + MgATP + CoASH → 2-hydroxybenzoyl-CoA + MgAMP + PP_i (2) 2-hydroxybenzoyl-CoA + 2[H] → benzoyl-CoA + H₂O Benzoyl-CoA was dearomatized by reduction of the ring. This represents another case in which benzoyl-CoA is a central intermediate in anaerobic aromatic metabolism. Received: 1 February 1996 / Accepted: 24 February 1996     

Proteogenomic evidence for β-oxidation of plant-derived 3-phenylpropanoids in "Aromatoleum aromaticum" EbN1

The betaproteobacterium "Aromatoleum aromaticum" EbN1 utilizes eight different plant-derived nonhydroxylated (e.g. cinnamate) and hydroxylated (e.g. p-coumarate) 3-phenylpropanoids with nitrate as electron acceptor. Differential protein profiling (2D-DIGE) revealed abundance increases of five proteins (EbA5316 to EbA5320) during anaerobic growth with cinnamate, hydrocinnamate, p-coumarate, and 3-(4-hydroxyphenyl)propanoate, compared to anaerobic benzoate-adapted cells serving as reference state. The predicted functions of four of these proteins (EbA5317, fatty acid-coenzyme A (CoA) ligase; EbA5318, enoyl-CoA hydratase/isomerase; EbA5319, β-ketothiolase; and EbA5320, 3-hydroxyacyl-CoA dehydrogenase) suggest β-oxidation of the above 3-phenylpropanoids to benzoyl-CoA and p-hydroxybenzoyl-CoA, respectively. The fifth protein (EbA5316, ABC-type periplasmic solute-binding protein) could be involved in 3-phenylpropanoid uptake. The detection of 3-hydroxy-3-phenylpropanoate during anaerobic growth with cinnamate and hydrocinnamate or 3-hydroxy-3-(4-hydroxyphenyl)propanoate during anaerobic growth with p-coumarate and 3-(4-hydroxyphenyl)propanoate supports the proteome-predicted β-oxidation pathway. Based on the specific formation of EbA5316-20 also during anaerobic growth with further 3-phenylpropanoid growth substrates including cinnamyl alcohol, m-coumarate, 3-(3,4-dihydroxyphenyl)propanoate and 3,4-dihydroxycinnamate (caffeate), a common β-oxidation route is proposed for 3-phenylpropanoid degradation in strain EbN1. The low amount of metabolites attributable to cometabolic transformation of nongrowth supporting 3-phenylpropanoids (e.g. o-coumarate, ferulate) may be indicative for a high substrate specificity of the involved enzymes.     

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.