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     

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.     

Anaerobic Metabolism of Catechol by the Denitrifying Bacterium Thauera aromatica—a Result of Promiscuous Enzymes and Regulators?

The anaerobic metabolism of catechol (1,2-dihydroxybenzene) was studied in the betaproteobacterium Thauera aromatica that was grown with CO₂ as a cosubstrate and nitrate as an electron acceptor. Based on different lines of evidence and on our knowledge of enzymes and genes involved in the anaerobic metabolism of other aromatic substrates, the following pathway is proposed. Catechol is converted to catechylphosphate by phenylphosphate synthase, which is followed by carboxylation by phenylphosphate carboxylase at the para position to the phosphorylated phenolic hydroxyl group. The product, protocatechuate (3,4-dihydroxybenzoate), is converted to its coenzyme A (CoA) thioester by 3-hydroxybenzoate-CoA ligase. Protocatechuyl-CoA is reductively dehydroxylated to 3-hydroxybenzoyl-CoA, possibly by 4-hydroxybenzoyl-CoA reductase. 3-Hydroxybenzoyl-CoA is further metabolized by reduction of the aromatic ring catalyzed by an ATP-driven benzoyl-CoA reductase. Hence, the promiscuity of several enzymes and regulatory proteins may be sufficient to create the catechol pathway that is made up of elements of phenol, 3-hydroxybenzoate, 4-hydroxybenzoate, and benzoate metabolism.     

Involvement of coenzyme A thioesters in anaerobic metabolism of 4-hydroxybenzoate by Rhodopseudomonas palustris.

The initial steps of anaerobic 4-hydroxybenzoate degradation were studied in whole cells and cell extracts of the photosynthetic bacterium Rhodopseudomonas palustris. Illuminated suspensions of cells that had been grown anaerobically on 4-hydroxybenzoate and were assayed under anaerobic conditions took up [U-14C]4-hydroxybenzoate at a rate of 0.6 nmol min-1 mg of protein-1. Uptake occurred with high affinity (apparent Km = 0.3 microM), was energy dependent, and was insensitive to external pH in the range of 6.5 to 8.2 Very little free 4-hydroxybenzoate was found associated with cells, but a range of intracellular products was formed after 20-s incubations of whole cells with labeled substrate. When anaerobic pulse-chase experiments were carried out with cells incubated on ice or in darkness, 4-hydroxybenzoyl coenzyme A (4-hydroxybenzoyl-CoA) was formed early and disappeared immediately after addition of excess unlabeled substrate, as would be expected of an early intermediate in 4-hydroxybenzoate metabolism. A 4-hydroxybenzoate-CoA ligase activity with an average specific activity of 0.7 nmol min-1 mg of protein-1 was measured in the soluble protein fraction of cells grown anaerobically on 4-hydroxybenzoate. 4-Hydroxybenzoyl-CoA was the sole product formed from labeled 4-hydroxybenzoate in the ligase reaction mixture. 4-Hydroxybenzoate uptake and ligase activities were present in cells grown anaerobically with benzoate, 4-hydroxybenzoate, and 4-aminobenzoate and were not detected in succinate-grown cells. These results indicate that the high-affinity uptake of 4-hydroxybenzoate by R. palustris is due to rapid conversion of the free acid to its CoA derivative by a CoA ligase and that this is also the initial step of anaerobic 4-hydroxybenzoate degradation.     

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.     

Aerobic and anaerobic catabolism of phthalic acid by a nitrate-respiring bacterium

A Bacillus sp., isolated by anaerobic enrichment on a o-phthalic acid-nitrate medium, grew either aerobically or anaerobically on phthalic acid. Cells grown anaerobically on phthalate immediately oxidized phthalate and benzoate with nitrate, whereas aerobic oxidation only occurred after a lag period and was inhibited by chloramphenicol. 2-Fluoro-and 3-fluorobenzoate were formed from 3-fluorophthalate by cells grown anaerobically on phthalate. Aerobically grown cells immediately oxidized phthalate, benzoate, 3-hydroxybenzoate and gentisate with oxygen. The aerobic and anaerobic route of catabolism of phthalate may thus share an initial decarboxylation to benzoate. This is the first report of the anaerobic dissimilation of phthalic acid by a pure bacterial culture.     

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.     

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.     

Reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA in a denitrifying, phenol-degrading Pseudomonas species

The initial reactions in anaerobic degradation of phenol to CO2 have been studied in vitro with a denitrifying Pseudomonas strain grown with phenol and nitrate in the absence of molecular oxygen. Phenol has been proposed to be carboxylated to 4-hydroxybenzoate [(1987) Arch. Microbiol. 148, 213-217]. 4-Hydroxybenzoate was activated to 4-hydroxybenzoyl-CoA by a coenzyme A ligase. Cell extracts also catalyzed the reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA with reduced benzyl viologen as electron donor. This enzyme, benzoyl-CoA:(acceptor) 4-oxidoreductase (hydroxylating) (EC 1.3.99.-), has not been reported before. The data suggest that phenol and 4-hydroxybenzoate are anaerobically metabolized by this strain via benzoyl-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.     

Purification and characterization of benzoate-coenzyme A ligase and 2-aminobenzoate-coenzyme A ligases from a denitrifying Pseudomonas sp.

The enzymes catalyzing the formation of coenzyme A (CoA) thioesters of benzoate and 2-aminobenzoate were studied in a denitrifying Pseudomonas sp. anaerobically grown with these aromatic acids and nitrate as sole carbon and energy sources. Three different rather specific aromatic acyl-CoA ligases, E1, E2, and E3, were found which catalyze the formation of CoA thioesters of benzoate, fluorobenzoates, and 2-aminobenzoate. ATP is cleaved into AMP and pyrophosphate. The enzymes were purified, their N-terminal amino acid sequences were determined, and their catalytic and molecular properties were studied. Cells anaerobically grown on benzoate and nitrate contain one CoA ligase (AMP forming) for benzoic acid (E1). It is a homodimer of Mr 120,000 which prefers benzoate as a substrate but shows some activity also with 2-aminobenzoate and fluorobenzoates, although with lower Km. Cells anaerobically grown on 2-aminobenzoate and nitrate contain three different CoA ligases for aromatic acids. The first one is identical with benzoate-CoA ligase (E1). The second enzyme is a 2-aminobenzoate-CoA ligase (E2). It is a monomer of Mr 60,000 which prefers 2-aminobenzoate but also activates benzoate, fluorobenzoates and, less effectively, 2-methylbenzoate, with lower affinities to the latter substrates. The enzymes E1 and E2 have similar activity levels; a third minor CoA ligase activity is due to a different 2-aminobenzoate-CoA ligase. The enzyme (E3) is a monomer of Mr, 65,000 which 2-aminobenzoate pathway (U. Altenschmidt, C. Eckerskorn, and G. Fuchs, Eur. J. Biochem. 194:647-653, 1990); apparently, it is not completely repressed under anaerobic conditions and therefore also is induced to a small extent by 2-aminobenzoate under anaerobic growth conditions.     

Anaerobic degradation of 3-hydroxybenzoate by a newly isolated nitrate-reducing bacterium

A Gram-negative nitrate-reducing bacterium, strain Asl-3, was isolated from activated sludge with nitrate and 3-hydroxybenzoate as sole source of carbon and energy. The new isolate was facultatively anaerobic, catalase- and oxidase-positive and polarly monotrichously flagellated. In addition to nitrate, nitrite, N2O, and O2 served as electron acceptors. Growth with 3-hydroxybenzoate and nitrate was biphasic: nitrate was completely reduced to nitrite before nitrite reduction to N2 started. Benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, protocatechuate or phenyl-acetate served as electron and carbon source under aerobic and anaerobic conditions. During growth with excess carbon source, poly-beta-hydroxybutyrate was formed. These characteristics allow the affiliation of strain Asl-3 with the family Pseudomonadaceae. Analogous to the pathway of 4-hydroxybenzoate degradation in other bacteria, the initial step in anaerobic 3-hydroxybenzoate degradation by this organism was activation to 3-hydroxy-benzoyl-CoA in an ATP-consuming reaction. Cell extracts of 3-hydroxybenzoate-grown cells exhibited 3-hydroxybenzoyl-CoA synthetase activity of 190 nmol min-1 mg protein-1 as well as benzoyl-CoA synthetase activity of 86 nmol min-1 mg protein-1. A reductive dehydroxylation of 3-hydroxybenzoyl-CoA could not be demonstrated due to rapid hydrolysis of chemically synthesized 3-hydroxybenzoyl-CoA by cell extracts.     

Activation of aromatic acids and aerobic 2-aminobenzoate metabolism in a denitrifying Pseudomonas strain

The initial reactions possibly involved in the acrobic and anaerobic metabolism of aromatic acids by a denitrifying Pseudomonas strain were studied. Several acyl CoA synthetases were found supporting the view that activation of several aromatic acids preceeds degradation. A benzoyl CoA synthetase activity (AMP forming) (apparent K _m values of the enzyme from nitrate grown cells: 0.01 mM benzoate, 0.2 mM ATP, 0.2 mM coenzyme A) was present in aerobically grown and anaerobically, nitrate grown cells when benzoate or other aromatic acids were present. In addition to benzoate and fluorobenzoates, also 2-amino-benzoate was activated, albeit with unfavorable K _m (0.5 mM 2-aminobenzoate). A 2-aminobenzoyl CoA synthetase (AMP forming) was induced both aerobically and anaerobically with 2-aminobenzoate as growth substrate which had a similar substrate spectrum but a low K _m for 2-aminobenzoate (<0.02 mM). Anaerobic growth on 4-hydroxybenzoate induced a 4-hydroxybenzoyl CoA synthetase, and cyclohexanecarboxylate induced another synthetase. In contrast, 3-hydroxybenzoate and phenyl-acetate grown anaerobic cells appeared not to activate the respective substrates at sufficient rates. Contrary to an earlier report extracts from aerobic and anaerobic 2-aminobenzoate grown cells catalysed a 2-aminobenzoyl CoA-dependent NADH oxidation. This activity was 10–20 times higher in aerobic cells and appeared to be induced by 2-aminobenzoate and oxygen. In vitro, 2-aminobenzoyl CoA reduction was dependent on 2-aminobenzoyl CoA NAD(P)H, and oxygen. A novel mechanism of aerobic 2-aminobenzoate degradation is suggested, which proceeds via 2-aminobenzoyl CoA.     

A novel pathway of aerobic benzoate catabolism in the bacteria Azoarcus evansii and Bacillus stearothermophilus

The aerobic catabolism of benzoate was studied in the Gram-negative proteobacterium Azoarcus evansii and in the Gram-positive bacterium Bacillus stearothermophilus. In contrast to earlier proposals, benzoate was not converted into hydroxybenzoate or gentisate. Rather, benzoyl-CoA was a product of benzoate catabolism in both microbial species under aerobic conditions in vivo. Benzoyl-CoA was converted into various CoA thioesters by cell extracts of both species in oxygen- and NADPH-dependent reactions. Using [ring-(13)C(6)]benzoyl-CoA as substrate, cis-3,4-[2,3,4,5,6-(13)C(5)]dehydroadipyl-CoA, trans-2,3-[2,3,4,5,6-(13)C(5)]dehydroadipyl-CoA, the 3,6-lactone of 3-[2,3,4,5,6-(13)C(5)]hydroxyadipyl-CoA, and 3-[2,3,4,5,6-(13)C(5)]hydroxyadipyl-CoA were identified as products by NMR spectroscopy. A protein mixture of A. evansii transformed [ring-(13)C(6)]benzoyl-CoA in an NADPH- and oxygen-dependent reaction into 6-[2,3,4,5,6-(13)C(5)]hydroxy-3-hexenoyl-CoA. The data suggest a novel aerobic pathway of benzoate catabolism via CoA intermediates leading to beta-ketoadipyl-CoA, an intermediate of the known beta-ketoadipate pathway.     

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.     

Anaerobic oxidation of phenylacetate and 4-hydroxyphenylacetate to benzoyl-coenzyme A and CO2 in denitrifying Pseudomonas sp.

Anaerobic degradation of (4-hydroxy)phenylacetate in denitrifying Pseudomonas sp. was investigated. Evidence is presented for -oxidation of the coenzyme A (CoA)-activated carboxymethyl side chain, a reaction which has not been described. The C₆–C₂ compounds are degraded to benzoyl-CoA and furtheron to CO₂ via the following intermediates: Phenylacetyl-CoA, phenylglyoxylate, benzoyl-CoA plus CO₂; 4-hydroxyphenylacetyl-CoA, 4-hydroxyphenylglyoxylate, 4-hydroxybenzoyl-CoA plus CO₂, benzoyl-CoA. Trace amounts of mandelate possibly derived from mandelyl-CoA were detected during phenylacetate degradation in vitro. The reactions are catalyzed by (i) phenylacetate-CoA ligase which converts phenylacetate to phenylacetyl-CoA and by a second enzyme for 4-hydroxyphenylacetate; (ii) a (4-hydroxy)-phenylacetyl-CoA dehydrogenase system which oxidizes phenylacetyl-CoA to (4-hydroxy)phenylglyoxylate plus CoA; and (iii) (4-hydroxy)phenylglyoxylate: acceptor oxidoreductase (CoA acylating) which catalyzes the oxidative decarboxylation of (4-hydroxy)phenylglyoxylate to (4-hydroxy)benzoyl-CoA and CO₂. (iv) The degradation of 4-hydroxyphenylacetate in addition requires the reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA, catalyzed by 4-hydroxybenzoyl-CoA reductase (dehydroxylating). The whole cell regulation of these enzyme activities supports the proposed pathway. An ionic mechanism for anaerobic -oxidation of the CoA-activated carboxymethyl side chain is proposed. Phenylacetic acids are plant constituents and in addition are formed from a large variety of natural aromatic compounds by microorganisms; their degradation therefore plays a significant role in nature, as illustrated in the preceding paper (Mohamed and Fuchs 1993). We have investigated and purified an enzyme which catalyzes the first step in the anaerobic degradation of phenylacetate in a denitrifying Pseudomonas sp. Phenylacetate is converted to phenylacetyl-CoA by phenylacetate-CoA ligase (AMP forming). The postulated function of this enzyme is corroborated by the strict regulation of its expression. 4-Hydroxyphenylacetate appears to be similarly activated by an independent enzyme prior to further degradation.We have suggested before that phenylacetyl-CoA is anaerobically converted by -oxidation of the side chain to phenylglyoxylate¹, which is oxidatively decarboxylated to benzoyl-CoA plus CO₂ (Seyfried et al. 1991; Dangel et al. 1991). 4-Hydroxyphenylacetate was proposed to be similarly oxidized to 4-hydroxybenzoyl-CoA plus CO₂, followed by reductive dehydroxylation to benzoyl-CoA. The evidence was not presented in full, and the crucial -oxidation was not demonstrated in vitro. We present here ample evidence for this pathway. A hypothetical mechanism is proposed by which the oxidation of the -methylene group to an -carbonyl group may occur.     

Anaerobic degradation of protocatechuate (3,4-dihydroxybenzoate) by Thauera aromatica strain AR-1

The denitrifying bacterium Thauera aromatica strain AR-1 grows anaerobically with protocatechuate (3,4-dihydroxybenzoate (DHB)) as sole energy and carbon source. This bacterium harbors two distinct pathways for degradation of aromatic compounds, the benzoyl-coenzyme A (CoA) pathway for benzoate degradation and the hydroxyhydroquinone (HHQ) pathway for degradation of 3,5-DHB. In order to elucidate whether protocatechuate is degraded via the benzoyl-CoA or the HHQ pathway, induction experiments were carried out. Dense suspensions of cells grown on protocatechuate or benzoate readily degraded benzoate and protocatechuate but not 3,5-DHB. Dense suspensions of 3,5-DHB-grown cells degraded 3,4- and 3,5-DHB at similar rates, but benzoate was not degraded. 3,5-DHB hydroxylating activity was found only in cells grown with this substrate. HHQ dehydrogenase activity was found in extracts of cells grown with 3,5-DHB and at a low rate also in protocatechuate-grown cells, but not in extracts of cells grown with benzoate. Activities of protocatechuyl-CoA synthetase and protocatechuyl-CoA reductase leading to 3-hydroxybenzoyl-CoA were found in extracts of cells grown with protocatechuate. There was no repression of the HHQ pathway by the presence of protocatechuate, unlike by degradation of benzoate. We conclude that protocatechuate is not degraded via the HHQ pathway because there was no evidence of a hydroxylation reaction involved in this process. Instead, our results strongly suggest that protocatechuate is degraded via a pathway which connects to the benzoyl-CoA route of degradation.     

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.