Purification and properties of 4-hydroxybenzoate 1-hydroxylase (decarboxylating), a novel flavin adenine dinucleotide-dependent monooxygenase from Candida parapsilosis CBS604.

A novel flavoprotein monooxygenase, 4-hydroxybenzoate 1-hydroxylase (decarboxylating), from Candida parapsilosis CBS604 was purified to apparent homogeneity. The enzyme is induced when the yeast is grown on either 4-hydroxybenzoate, 2,4-dihydroxybenzoate, or 3,4-dihydroxybenzoate as the sole carbon source. The purified monooxygenase is a monomer of about 50 kDa containing flavin adenine dinucleotide as weakly bound cofactor. 4-Hydroxybenzoate 1-hydroxylase from C. parapsilosis catalyzes the oxidative decarboxylation of a wide range of 4-hydroxybenzoate derivatives with the stoichiometric consumption of NAD(P)H and oxygen. Optimal catalysis is reached at pH 8, with NADH being the preferred electron donor. By using (18)O2, it was confirmed that the oxygen atom inserted into the product 1,4-dihydroxybenzene is derived from molecular oxygen. 19F nuclear magnetic resonance spectroscopy revealed that the enzyme catalyzes the conversion of fluorinated 4-hydroxybenzoates to the corresponding hydroquinones. The activity of the enzyme is strongly inhibited by 3,5-dichloro-4-hydroxybenzoate, 4-hydroxy-3,5-dinitrobenzoate, and 4-hydroxyisophthalate, which are competitors with the aromatic substrate. The same type of inhibition is exhibited by chloride ions. Molecular orbital calculations show that upon deprotonation of the 4-hydroxy group, nucleophilic reactivity is located in all substrates at the C-1 position. This, and the fact that the enzyme is highly active with tetrafluoro-4-hydroxybenzoate and 4-hydroxy-3-nitrobenzoate, suggests that the phenolate forms of the substrates play an important role in catalysis. Based on the substrate specificity, a mechanism is proposed for the flavin-mediated oxidative decarboxylation of 4-hydroxybenzoate.     

Purification and properties of hydroquinone hydroxylase, a FAD-dependent monooxygenase involved in the catabolism of 4-hydroxybenzoate in Candida parapsilosis CBS604.

The ascomycetous yeast Candida parapsilosis CBS604 catabolizes 4-hydroxybenzoate through the initial formation of hydroquinone (1, 4-dihydroxybenzene). High levels of hydroquinone hydroxylase activity are induced when the yeast is grown on either 4-hydroxybenzoate, 2,4-dihydroxybenzoate, 1,3-dihydroxybenzene or 1, 4-dihydroxybenzene as the sole carbon source. The monooxygenase constitutes up to 5% of the total amount of protein and is purified to apparent homogeneity in three chromatographic steps. Hydroquinone hydroxylase from C. parapsilosis is a homodimer of about 150 kDa with each 76-kDa subunit containing a tightly noncovalently bound FAD. The flavin prosthetic group is quantitatively resolved from the protein at neutral pH in the presence of chaotropic salts. The apoenzyme is dimeric and readily reconstituted with FAD. Hydroquinone hydroxylase from C. parapsilosis catalyzes the ortho-hydroxylation of a wide range of monocyclic phenols with the stoichiometric consumption of NADPH and oxygen. With most aromatic substrates, no uncoupling of hydroxylation occurs. Hydroxylation of monofluorinated phenols is highly regiospecific with a preference for C6 hydroxylation. Binding of phenol highly stimulates the rate of flavin reduction by NADPH. At pH 7.6, 25 degrees C, this step does not limit the rate of overall catalysis. During purification, hydroquinone hydroxylase is susceptible towards limited proteolysis. Proteolytic cleavage does not influence the enzyme dimeric nature but results in relatively stable protein fragments of 55, 43, 35 and 22 kDa. N-Terminal peptide sequence analysis revealed the presence of two nick sites and showed that hydroquinone hydroxylase from C. parapsilosis is structurally related to phenol hydroxylase from Trichosporon cutaneum. The implications of these findings for the catalytic mechanism of hydroquinone hydroxylase are discussed.     

Purification, characterization, and gene cloning of 4-hydroxybenzoate decarboxylase of Enterobacter cloacae P240

We found the occurrence of 4-hydroxybenzoate decarboxylase in Enterobacter cloacae P240, isolated from soils under anaerobic conditions, and purified the enzyme to homogeneity. The purified enzyme was a homohexamer of identical 60 kDa subunits. The purified decarboxylase catalyzed the nonoxidative decarboxylation of 4-hydroxybenzoate without requiring any cofactors. Its K _m value for 4-hydroxybenzoate was 596 μM. The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate, for which the K _m value was 6.80 mM. In the presence of 3 M KHCO₃ and 20 mM phenol, the decarboxylase catalyzed the reverse carboxylation reaction of phenol to form 4-hydroxybenzoate with a molar conversion yield of 19%. The K _m value for phenol was calculated to be 14.8 mM. The gene encoding the 4-hydroxybenzoate decarboxylase was isolated from E. cloacae P240. Nucleotide sequencing of recombinant plasmids revealed that the 4-hydroxybenzoate decarboxylase gene codes for a 475-amino-acid protein. The amino acid sequence of the enzyme is similar to those of 4-hydroxybenzoate decarboxylase of Clostridium hydroxybenzoicum (53% identity), VdcC protein (vanillate decarboxylase) of Streptomyces sp. strain D7 (72%) and 3-octaprenyl-4-hydroxybenzoate decarboxylase of Escherichia coli (28%). The hypothetical proteins, showing 96–97% identities to the primary structure of E. cloacae P240 4-hydroxybenzoate decarboxylase, were found in several bacterial strains.     

Anaerobic degradation of 4-hydroxybenzoate: Reductive dehydroxylation of 4-hydroxybenzoyl-CoA and ATP formation during 4-hydroxybenzoate decarboxylation by the phenol-metabolizing bacteria of a stable,strictly anaerobic consortium

4-Hydroxybenzoate was activated with coenzyme A by cells of a strictly anaerobic, phenol-degrading mixed culture to 4-hydroxybenzoyl-CoA, which was reductively dehydroxylated to benzoyl-CoA with reduced benzylviologen as an electron donor. The specific activity of the 4-hydroxybenzoyl-CoA ligase in cell-free extracts of the culture was 100–200 nmol min^–1 mg^–1, that of 4-hydroxybenzoyl-CoA reductase 14.5 nmol min^–1 mg^–1. An increased growth yield of the phenol-degrading mixed culture of 1.8 g/mol with 4-hydroxybenzoate in comparison to phenol as the substrate was found previously and indicated energy generation by decarboxylation of 4-hydroxybenzoate. Addition of 4-hydroxybenzoate to cell suspensions of the mixed culture resulted in a rapid increase of the cellular ATP level. The proton ionophore carbonylcyanidem-chlorophenylhydrazone and the H⁺-ATPase inhibitor dicyclohexylcarbodiimide prevented an increase of cellular ATP levels during 4-hydroxybenzoate decarboxylation, whereas the sodium ionophore monensin and the putative Na⁺-ATPase inhibitor ouabain revealed no effect. This was taken as good evidence for the generation of a proton gradient across the membrane by decarboxylation of 4-hydroxybenzoate and ATP formation by H+-ATPase.     

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 degradation of phenol via carboxylation to 4-hydroxybenzoate: in vitro study of isotope exchange between 14CO2 and 4-hydroxybenzoate

Extracts of denitrifying bacteria grown anaerobically with phenol and nitrate catalyzed an isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate. This exchange reaction is ascribed to a novel enzyme, phenol carboxylase, initiating the anaerobic degradation of phenol by para-carboxylation to 4-hydroxybenzoate. Some properties of this enzyme were determined by studying the isotope exchange reaction. Phenol carboxylase was rapidly inactivated by oxygen; strictly anoxic conditions were essential for preserving enzyme activity. The exchange reaction specifically was catalyzed with 4-hydroxybenzoate but not with other aromatic acids. Only the carboxyl group was exchanged; [U-¹⁴C]phenol was not exchanged with the aromatic ring of 4-hydroxybenzoate. Exchange activity depended on Mn²⁺ and inorganic phosphate and was not inhibited by avidin. Ortho-phosphate could not be substituted by organic phosphates nor by inorganic anions; arsenate had no effect. The pH optimum was between pH 6.5–7.0. The specific activity was 100 nmol ¹⁴CO₂ exchange · min^-1 · mg^-1 protein. Phenol grown cells contained 4-hydroxybenzoyl CoA synthetase activity (40 nmol · min^-1 · mg^-1 protein). The possible role of phenol carboxylase and 4-hydroxybenzoyl CoA synthetase in anaerobic phenol metabolism is discussed.     

Pathways of 4-hydroxybenzoate degradation among species of Bacillus.

The pathways used by three bacterial strains of the genus Bacillus to degrade 4-hydroxybenzoate are delineated. When B. brevis strain PHB-2 is grown on 4-hydroxybenzoate, enzymes of the protocatechuate branch of the beta-ketoadipate pathway are induced. In contrast, B. circulans strain 3 contains high levels of the enzymes of the protocatechuate 2,3-dioxygenase pathway after growth on 4-hydroxybenzoate. B. laterosporus strain PHB-7a degrades 4-hydroxybenzoate by a novel reaction sequence. After growth on 4-hydroxybenzoate, strain PHB-7a contains high levels of gentisate oxygenase (EC 1.13.11.4) and maleylpyruvate hydrolase. Whole cells of strain PHB-7a (grown on 4-hydroxylbenzoate) accumulate 2,5-dihydroxybenzoate (gentisate) from 4-hydroxybenzoate when incubated in the presence of 1mM alpha,alpha'-dipyridyl. Thus, strain PHB-7a appears to convert 4-hydroxybenzoate to gentisate, which is further degraded by the glutathione-independent gentisic acid pathway. These pathway delineations provide evidence that Bacillus species are derived from a diverse evolutionary background.     

Regioselective carboxylation of 1,3-dihydroxybenzene by 2,6-dihydroxybenzoate decarboxylase of Pandoraea sp. 12B-2

We found a bacterium, Pandoraea sp. 12B-2, of which whole cells catalyzed not only the decarboxylation of 2,6-dihydroxybenzoate but also the regioselective carboxylation of 1,3-dihydroxybenzene to 2,6-dihydroxybenzoate. The whole cells of Pandoraea sp. 12B-2 also catalyzed the regioselective carboxylation of phenol and 1,2-dihydroxybenzene to 4-hydroxybenzoate and 2,3-dihydroxybenzoate, respectively. The molar conversion ratio of the carboxylation reaction depended on the concentration of KHCO₃ in the reaction mixture. Only 5 or 48 % of 1,3-dihydroxybenzene added was converted into 2,6-dihydroxybenzoate in the presence of 0.1 M or 3 M KHCO₃, respectively. The addition of acetone to the reaction mixture increased the initial rate of the carboxylation reaction, but the final molar conversion yield reached almost the same value. When the efficient production of 2,6-dihydroxybenzoate was optimized using the whole cells of Pandoraea sp. 12B-2, the productivity of 2,6-dihydroxybenzoate topped out at 1.43 M, which was the highest value so far reported. No formation of any other products was observed after the carboxylation reaction.     

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 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.     

Degradation of 2,7-dichlorodibenzo-p-dioxin by the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Under secondary metabolic conditions, the white-rot basidiomycete Phanerochaete chrysosporium degraded 2,7-dichlorodibenzo-p-dioxin (I). The pathway for the degradation of I was elucidated by the characterization of fungal metabolites and oxidation products generated by lignin peroxidase (LiP), manganese peroxidase (MnP), and crude intracellular cell-free extracts. The multistep pathway involves the degradation of I and subsequent intermediates by oxidation, reduction, and methylation reactions to yield the key intermediate 1,2,4-trihydroxybenzene (III). In the first step, the oxidative cleavage of the dioxin ring of I, catalyzed by LiP, generates 4-chloro-1,2-benzoquinone (V), 2-hydroxy-1,4-benzoquinone (VIII), and chloride. The intermediate V is then reduced to 1-chloro-3,4-dihydroxybenzene (II), and the latter is methylated to form 1-chloro-3,4-dimethoxybenzene (VI). VI in turn is oxidized by LiP to generate chloride and 2-methoxy-1,4-benzoquinone (VII), which is reduced to 2-methoxy-1,4-dihydroxybenzene (IV). IV is oxidized by either LiP or MnP to generate 4-hydroxy-1,2-benzoquinone, which is reduced to 1,2,4-trihydroxybenzene (III). The other aromatic product generated by the initial LiP-catalyzed cleavage of I is 2-hydroxy-1,4-benzoquinone (VIII). This intermediate is also generated during the LiP- or MnP-catalyzed oxidation of the intermediate chlorocatechol (II). VIII is also reduced to 1,2,4-trihydroxybenzene (III). The key intermediate III is ring cleaved by intracellular cell extracts to produce, after reduction, beta-ketoadipic acid. In this pathway, initial oxidative cleavage of both C-O-C bonds in I by LiP generates two quinone products, 4-chloro-1,2-benzoquinone (V) and 2-hydroxy-1,4-benzoquinone (VIII). The former is recycled by reduction and methylation reactions to generate an intermediate which is also a substrate for peroxidase-catalyzed oxidation, leading to the removal of a second chlorine atom. This unique pathway results in the removal of both aromatic chlorines before aromatic ring cleavage takes place.     

PcaK, a high-affinity permease for the aromatic compounds 4-hydroxybenzoate and protocatechuate from Pseudomonas putida.

PcaK is a transporter and chemoreceptor protein from Pseudomonas putida that is encoded as part of the beta-ketoadipate pathway regulon for aromatic acid degradation. When expressed in Escherichia coli, PcaK was localized to the membrane and catalyzed the accumulation of two aromatic substrates, 4-hydroxybenzoate and protocatechuate, against a concentration gradient. Benzoate inhibited 4-hydroxybenzoate uptake but was not a substrate for PcaK-catalyzed transport. A P. putida pcaK mutant was defective in its ability to accumulate micromolar amounts of 4-hydroxybenzoate and protocatechuate. The mutant was also impaired in growth on millimolar concentrations of these aromatic acids. In contrast, the pcaK mutant grew at wild-type rates on benzoate. The Vmax for uptake of 4-hydroxybenzoate was at least 25 nmol/min/mg of protein, and the Km was 6 microM. PcaK-mediated transport is energized by the proton motive force. These results show that although aromatic acids in the undissociated (uncharged) form can diffuse across bacterial membranes, high-specificity active transport systems probably also contribute to the ability of bacteria to grow on the micromolar concentrations of these compounds that are typically present in soil. A variety of aromatic molecules, including naturally occurring lignin derivatives and xenobiotics, are metabolized by bacteria and may be substrates for transport proteins. The characterization of PcaK provides a foundation for understanding active transport as a critical step in the metabolism of aromatic carbon sources.     

Identification of the pcaRKF gene cluster from Pseudomonas putida: involvement in chemotaxis, biodegradation, and transport of 4-hydroxybenzoate.

Pseudomonas putida PRS2000 is chemotactic to 4-hydroxybenzoate and other aromatic acids. This behavioral response is induced when cells are grown on 4-hydroxybenzoate or benzoate, compounds that are degraded via the beta-ketoadipate pathway. Isolation of a transposon mutant defective in 4-hydroxybenzoate chemotaxis allowed identification of a new gene cluster designated pcaRKF. DNA sequencing, mutational analysis, and complementation studies revealed that pcaR encodes a regulatory protein required for induction of at least four of the enzymes of the beta-ketoadipate pathway and that pcaF encodes beta-ketoadipyl-coenzyme A thiolase, the last enzyme in the pathway. The third gene, pcaK, encodes a transporter for 4-hydroxybenzoate, and this protein is also required for chemotaxis to aromatic acids. The predicted PcaK protein is 47 kDa in size, with a deduced amino acid sequence indicative of membership in the major facilitator superfamily of transport proteins. The protein, expressed in Escherichia coli, catalyzed 4-hydroxybenzoate transport. In addition, whole cells of P. putida pcaK mutants accumulated 4-hydroxybenzoate at reduced rates compared with that in wild-type cells. The pcaK mutation did not impair growth at the expense of 4-hydroxybenzoate under most conditions; however, mutant cells grew somewhat more slowly than the wild type on 4-hydroxybenzoate at a high pH. The finding that 4-hydroxybenzoate chemotaxis can be disrupted without an accompanying effect on metabolism indicates that this chemotactic response is receptor mediated. It remains to be determined, however, whether PcaK itself is a chemoreceptor for 4-hydroxybenzoate or whether it plays an indirect role in chemotaxis. These findings indicate that aromatic acid detection and transport are integral features of aromatic degradation pathways.     

Anaerobic degradation ofm-cresol via methyl oxidation to 3-hydroxybenzoate by a denitrifying bacterium

The anaerobic degradation of m-cresol was studied in a denitrifying bacterium. In the initial studies, hypothetical intermediates of m-cresol degradation were tested in growth experiments and in adaptation studies with dense cell suspensions. Results suggested a degradation of m-cresol via 3-hydroxybenzoate. To verify this, the degradation of m-cresol was followed in concentrated cell suspensions in the presence of metabolic inhibitors. Fluoroacetate treatment resulted in the transient accumulation of substantial amounts of 3-hydroxybenzoate. In the presence of iodoacetamide, not only was 3-hydroxybenzoate transiently formed, but benzoate was also accumulated. These findings support a degradation of m-cresol via initial anaerobic methyl oxidation to 3-hydroxybenzoate, followed by reductive dehydroxylation to benzoate or benzoyl-CoA. Studies with extracts of m-cresol-grown cells showed the presence of several enzyme activities to be postulated for this pathway. No evidence was found for a carboxylation, hydroxylation of the aromatic ring, or direct ring reduction as the initial step in m-cresol metabolism. Received: 29 November 1994 / Accepted: 7 March 1995     

Reductive dechlorination of 2,4-dichlorobenzoate to 4-chlorobenzoate and hydrolytic dehalogenation of 4-chloro-, 4-bromo-, and 4-iodobenzoate by Alcaligenes denitrificans NTB-1

Alcaligenes denitrificans NTB-1, previously isolated on 4-chlorobenzoate, also utilized 4-bromo-, 4-iodo-, and 2,4-dichlorobenzoate but not 4-fluorobenzoate as a sole carbon and energy source. During growth, stoichiometric amounts of halide were released. Experiments with whole cells and cell extracts revealed that 4-bromo- and 4-iodobenzoate were metabolized like 4-chlorobenzoate, involving an initial hydrolytic dehalogenation yielding 4-hydroxybenzoate, which in turn was hydroxylated to 3,4-dihydroxybenzoate. The initial step in the metabolism of 2,4-dichlorobenzoate was catalyzed by a novel type of reaction for aerobic organisms, involving inducible reductive dechlorination to 4-chlorobenzoate. Under conditions of low and controlled oxygen concentrations, A. denitrificans NTB-1 converted all 4-halobenzoates and 2,4-dichlorobenzoate almost quantitatively to 4-hydroxybenzoate.     

Reductive dechlorination of 2,4-dichlorobenzoate to 4-chlorobenzoate and hydrolytic dehalogenation of 4-chloro-, 4-bromo-, and 4-iodobenzoate by Alcaligenes denitrificans NTB-1.

Alcaligenes denitrificans NTB-1, previously isolated on 4-chlorobenzoate, also utilized 4-bromo-, 4-iodo-, and 2,4-dichlorobenzoate but not 4-fluorobenzoate as a sole carbon and energy source. During growth, stoichiometric amounts of halide were released. Experiments with whole cells and cell extracts revealed that 4-bromo- and 4-iodobenzoate were metabolized like 4-chlorobenzoate, involving an initial hydrolytic dehalogenation yielding 4-hydroxybenzoate, which in turn was hydroxylated to 3,4-dihydroxybenzoate. The initial step in the metabolism of 2,4-dichlorobenzoate was catalyzed by a novel type of reaction for aerobic organisms, involving inducible reductive dechlorination to 4-chlorobenzoate. Under conditions of low and controlled oxygen concentrations, A. denitrificans NTB-1 converted all 4-halobenzoates and 2,4-dichlorobenzoate almost quantitatively to 4-hydroxybenzoate.     

Regulation of the utilization of 4-hydroxybenzoate and 4-hydroxycinnamate in batch and continuous cultures of Pseudomonas testosteroni

Pseudomonas testosteroni metabolized 4-hydroxycinnamate by an initial cleavage of the side chain to yield acetate and the aromatic moiety, 4-hydroxybenzaldehyde. The latter was further oxidized via 4-hydroxybenzoate to protocatechuate, which underwent meta cleavage. During growth of the organism on 4-hydroxycinnamate, the for acetate showed an undulating pattern, which was attributed to alternating induction and repression of enzymes involved in the oxidation of acetate. Repression was caused either by 4-hydroxybenzoate or by its later metabolites, formate and pyruvate.In batch culture, P. testosteroni oxidized mixtures of 4-hydroxybenzoate and 4-hydroxycinnamate in a diauxic pattern. The capacity to oxidize 4-hydroxycinnamate appeared in the cells before 4-hydroxybenzoate was exhausted, indicating that the enzymes catalysing the conversion of 4-hydroxycinnamate into 4-hydroxybenzoate. were induced despite the presence of 4-hydroxybenzoate. The induction of these early enzymes of 4-hydroxycinnamate catabolism started when the molar concentration ratio of 4-hydroxybenzoate to 4-hydroxycinnamate fell below a value of 0.3.In continuous culture of P. testosteroni on a mixture of 4-hydroxybenzoate and 4-hydroxycinnamate, both substrates were almost completely utilized up to a dilution rate of about 0.5/h. At higher dilution rates, 4-hydroxycinnamate was decreasingly utilized so that eventually at a dilution rate of 0.74/h, its effluent concentration equalled its influent concentration. At D _M, a utilization ratio of 1.23 in favour of 4-hydroxybenzoate was found to become established in the culture. The of the cells for acetate was maximal at a dilution rate of 0.38/h and decreased before 4-hydroxycinnamate utilization was at its peak at 0.59/h. This suggested that it was mainly the aromatic moiety of 4-hydroxycinnamate which was metabolized at high dilution rates. The failure to utilize acetate at high dilution rates was apparently due to the repression of its catabolic enzymes by later metabolites of 4-hydroxybenzoate and to the relatively low concentration of acetate in the fermenter. This low concentration, due to the continuous washout of acetate, prevented it from relieving the repression.Abbreviations 4HB 4-hydroxybenzoate - 4HC 4-hydroxycinnamate - D _M dilution rate allowing maximal cell output rate - OD optical density     

Regulation of the utilization of 4-hydroxybenzoate and vanillate in batch and continuous cultures of Pseudomonas acidovorans

In Pseudomonas acidovorans, the pathways of 4-hydroxybenzoate and vanillate metabolism converge on the early intermediate, protocatechuate, which undergoes meta-cleavage. The methoxyl group of vanillate is almost completely oxidized, as shown by an experiment with (¹⁴C-methoxyl) vanillate. In batch cultures, 4-hydroxybenzoate and vanillate are simultaneously oxidized. Simultaneous oxidation was explained above all by the fact that both substrates mutually repress the ability of the cells to utilize the partner substrate.If P. acidovorans is growing in a turbidostat on one of the two substrates and is suddenly exposed to an equimolar mixture of both substrates, the respiration rates for the two substrates reciprocate, the for the substrate utilized first passing through a transient minimum, that for the added substrate passing through a transient maximum. Finally, a balance appears to be established, the for 4-hydroxybenzoate being slightly above that for vanillate. Transient phenomena also occur if a chemostat culture with both substrates is suddenly operated as a turbidostat culture or if cells not adapted to either substrate are suddenly exposed to a mixture of both substrates in the turbidostat.If a chemostat culture of P. acidovorans, growing at the expense of an equimolar mixture of 4-hydroxybenzoate and vanillate, is operated under conditions of increasing oxygen deficiency, the utilization ratio of the two substrates increases in favour of 4-hydroxybenzoate. However, if the culture is operated under conditions of increasing nitrogen deficiency, the utilization ratio increases in favour of vanillate.Abbreviations 4HB 4-hydroxybenzoate - VA vanillate - OD optical density     

Catalytic properties of phenol carboxylase. In vitro study of CO2: 4-hydroxybenzoate isotope exchange reaction

Phenol is metabolized in a denitrifying bacterium in the absence of molecular oxygen via para-carboxylation to 4-hydroxybenzoate (biological Kolbe-Schmitt synthesis). The enzyme system catalyzing the presumptive carboxylation of phenol, tentatively named 'phenol carboxylase', catalyzes an isotope exchange between 14CO2 and the carboxyl group of 4-hydroxybenzoate (specific activity 0.1 mumol 14CO2 incorporated into 4-hydroxybenzoate x min-1 x mg-1 cell protein) which is considered a partial reaction of the overall enzyme catalysis; 14C from [14C]phenol was not exchanged into 4-hydroxybenzoate ring positions to a significant extent. The 14CO2 isotope exchange reaction was studied in vitro. The reaction was dependent on the substrates CO2 and 4-hydroxybenzoate and required K+ and Mn2+. The actual substrate was CO2 rather than HCO3-. The apparent Km values were 1 mM dissolved CO2, 0.2 mM 4-hydroxybenzoate, 2 mM K+, and 0.1 mM Mn2+. The cationic cocatalysts could be substituted by ions of similar ionic radius: K+ could be replaced to some extent by Rb+, but not by Li+, Na+, Cs+, or NH4+; Mn2+ could be replaced to some extent by Fe2+ greater than Mg2+, Co2+, but not by Ni2+, Zn2+, Ca2+, or Cu2+. The exchange reaction was not strictly specific for 4-hydroxybenzoate, however it required a p-hydroxyl group; derivatives of 4-hydroxybenzoate with OH, CH3 or Cl substituents in m-position did react, whereas those with substitutions in the o-position were inactive or were inhibitory. The enzyme was induced when cells were grown on phenol, but not on 4-hydroxybenzoate. Comparison of SDS/PAGE protein patterns of cells grown on phenol or 4-hydroxybenzoate revealed several additional protein bands in phenol-grown cells. The possible role of similar enzymes in the anaerobic metabolism of phenolic compounds is discussed.     

4-hydroxybenzoyl coenzyme A reductase (dehydroxylating) is required for anaerobic degradation of 4-hydroxybenzoate by Rhodopseudomonas palustris and shares features with molybdenum-containing hydroxylases.

The anaerobic degradation of 4-hydroxybenzoate is initiated by the formation of 4-hydroxybenzoyl coenzyme A, with the next step proposed to be a dehydroxylation to benzoyl coenzyme A, the starting compound for a central pathway of aromatic compound ring reduction and cleavage. Three open reading frames, divergently transcribed from the 4-hydroxybenzoate coenzyme A ligase gene, hbaA, were identified and sequenced from the phototrophic bacterium Rhodopseudomonas palustris. These genes, named hbaBCD, specify polypeptides of 17.5, 82.6, and 34.5 kDa, respectively. The deduced amino acid sequences show considerable similarities to a group of hydroxylating enzymes involved in CO, xanthine, and nicotine metabolism that have conserved binding sites for [2Fe-2S] clusters and a molybdenum cofactor. Cassette disruption of the hbaB gene yielded a mutant that was unable to grow anaerobically on 4-hydroxybenzoate but grew normally on benzoate. The hbaB mutant cells did not accumulate [14C]benzoyl coenzyme A during short-term uptake of [14C]4-hydroxybenzoate, but benzoyl coenzyme A was the major radioactive metabolite formed by the wild type. In addition, crude extracts of the mutant failed to convert 4-hydroxybenzoyl coenzyme A to benzoyl coenzyme A. This evidence indicates that the hbaBCD genes encode the subunits of a 4-hydroxybenzoyl coenzyme A reductase (dehydroxylating). The sizes of the specified polypeptides are similar to those reported for 4-hydroxybenzoyl coenzyme A reductase isolated from the denitrifying bacterium Thauera aromatica. The amino acid consensus sequence for a molybdenum cofactor binding site is in HbaC. This cofactor appears to be an essential component because anaerobic growth of R. palustris on 4-hydroxybenzoate, but not on benzoate, was retarded unless 0.1 microM molybdate was added to the medium. Neither tungstate nor vanadate replaced molybdate, and tungstate competitively inhibited growth stimulation by molybdate.