Reductive, Coenzyme A-Mediated Pathway for 3-Chlorobenzoate Degradation in the Phototrophic Bacterium Rhodopseudomonas palustris

We isolated a strain of Rhodopseudomonas palustris (RCB100) by selective enrichment in light on 3-chlorobenzoate to investigate the steps that it uses to accomplish anaerobic dechlorination. Analyses of metabolite pools as well as enzyme assays suggest that R. palustris grows on 3-chlorobenzoate by (i) converting it to 3-chlorobenzoyl coenzyme A (3-chlorobenzoyl–CoA), (ii) reductively dehalogenating 3-chlorobenzoyl–CoA to benzoyl-CoA, and (iii) degrading benzoyl-CoA to acetyl-CoA and carbon dioxide. R. palustris uses 3-chlorobenzoate only as a carbon source and thus incorporates the acetyl-CoA that is produced into cell material. The reductive dechlorination route used by R. palustris for 3-chlorobenzoate degradation differs from those previously described in that a CoA thioester, rather than an unmodified aromatic acid, is the substrate for complete dehalogenation.     

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.     

The Catalytic Mechanism of the Hotdog-fold Enzyme Superfamily 4-Hydroxybenzoyl-CoA Thioesterase from Arthrobacter sp. Strain SU

The hotdog-fold enzyme 4-hydroxybenzoyl-coenzyme A (4-HB-CoA) thioesterase from Arthrobacter sp. strain AU catalyzes the hydrolysis of 4-HB-CoA to form 4-hydroxybenzoate (4-HB) and coenzyme A (CoA) in the final step of the 4-chlorobenzoate dehalogenation pathway. Guided by the published X-ray structures of the liganded enzyme (Thoden, J. B., Zhuang, Z., Dunaway-Mariano, D., and Holden H. M. (2003) J.Biol. Chem. 278, 43709-43716), a series of site-directed mutants were prepared for testing the roles of active site residues in substrate binding and catalysis. The mutant thioesterases were subjected to X-ray structure determination to confirm retention of the native fold, and in some cases, to reveal changes in the active site configuration. In parallel, the wild-type and mutant thioesterases were subjected to transient and steady-state kinetic analysis, and to (18)O-solvent labeling experiments. Evidence is provided that suggests that Glu73 functions in nucleophilic catalysis, that Gly65 and Gln58 contribute to transition-state stabilization via hydrogen bond formation with the thioester moiety and that Thr77 orients the water nucleophile for attack at the 4-hydroxybenzoyl carbon of the enzyme-anhydride intermediate. The replacement of Glu73 with Asp was shown to switch the function of the carboxylate residue from nucleophilic catalysis to base catalysis and thus, the reaction from a two-step process involving a covalent enzyme intermediate to a single-step hydrolysis reaction. The E73D/T77A double mutant regained most of the catalytic efficiency lost in the E73D single mutant. The results from (31)P NMR experiments indicate that the substrate nucleotide unit is bound to the enzyme surface. Kinetic analysis of site-directed mutants was carried out to determine the contributions made by Arg102, Arg150, Ser120, and Thr121 in binding the nucleotide unit. Lastly, we show by kinetic and X-ray analyses of Asp31, His64, and Glu78 site-directed mutants that these three active site residues are important for productive binding of the substrate 4-hydroxybenzoyl ring.     

The purification and characterisation of 4-chlorobenzoate:CoA ligase and 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain TM-1

4-Chlorobenzoate:CoA ligase, the first enzyme in the pathway for 4-chlorobenzoate dissimilation, has been partially purified from Arthrobacter sp. strain TM-1, by sequential ammonium sulphate precipitation and chromatography on DEAE-Sepharose and Sephacryl S-200. The enzyme, a homodimer of subunit molecular mass approximately 56 kD, is dependent on Mg2+-ATP and coenzyme A, and produces 4-chlorobenzoyl CoA and AMP. Besides Mg2+, Mn2+, Co2+, Fe2+ and Zn2+ are also stimulatory, but not Ca2+. Maximal activity is exhibited at pH 7.0 and 25 degrees C. The ligase demonstrates broad specificity towards other halobenzoates, with 4-chlorobenzoate as best substrate. The apparent Michaelis constants (Km) of the enzyme for 4-chlorobenzoate, CoA and ATP were determined as 3.5, 30 and 238 microM respectively. 4-Chlorobenzoyl CoA dehalogenase, the second enzyme, has been purified to homogeneity by sequential column chromatography on hydroxyapatite, DEAE-Sepharose and Sephacryl S-200. It is a homotetramer of 33 kD subunits with an isoelectric point of 6.4. At pH 7.5 and 30 degrees C, Km and kcat for 4-CBCoA are 9 microM and 1 s(-1) respectively. The optimum pH is 7.5, and maximal enzymic activity occurs at 45 degrees C. The properties of this enzyme are compared with those of the 4-chlorobenzoyl CoA dehalogenases from Arthrobacter sp. strain 4-CB1 and Pseudomonas sp. strain CBS-3, which differ variously in their N-terminal amino acid sequences, optimal pH values, pI values and/or temperatures of maximal activity.     

Investigation of the catalytic mechanism of the hotdog-fold enzyme superfamily Pseudomonas sp. strain CBS3 4-hydroxybenzoyl-CoA thioesterase

The 4-hydroxybenzoyl-CoA (4-HB-CoA) thioesterase from Pseudomonas sp. strain CBS3 catalyzes the final step of the 4-chlorobenzoate degradation pathway, which is the hydrolysis of 4-HB-CoA to coenzyme A (CoA) and 4-hydroxybenzoate (4-HB). In previous work, X-ray structural analysis of the substrate-bound thioesterase provided evidence of the role of an active site Asp17 in nucleophilic catalysis [Thoden, J. B., Holden, H. M., Zhuang, Z., and Dunaway-Mariano, D. (2002) X-ray crystallographic analyses of inhibitor and substrate complexes of wild-type and mutant 4-hydroxybenzoyl-CoA thioesterase. J. Biol. Chem. 277, 27468-27476]. In the study presented here, kinetic techniques were used to test the catalytic mechanism that was suggested by the X-ray structural data. The time course for the multiple-turnover reaction of 50 μM [(14)C]-4-HB-CoA catalyzed by 10 μM thioesterase supported a two-step pathway in which the second step is rate-limiting. Steady-state product inhibition studies revealed that binding of CoA (K(is) = 250 ± 70 μM; K(ii) = 900 ± 300 μM) and 4-HB (K(is) = 1.2 ± 0.2 mM) is weak, suggesting that product release is not rate-limiting. A substantial D(2)O solvent kinetic isotope effect (3.8) on the steady-state k(cat) value (18 s(-1)) provided evidence that a chemical step involving proton transfer is the rate-limiting step. Taken together, the kinetic results support a two-chemical pathway. The microscopic rate constants governing the formation and consumption of the putative aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate were determined by simulation-based fitting of a kinetic model to time courses for the substrate binding reaction (5.0 μM 4-HB-CoA and 0.54 μM thioesterase), single-turnover reaction (5 μM [(14)C]-4-HB-CoA catalyzed by 50 μM thioesterase), steady-state reaction (5.2 μM 4-HB-CoA catalyzed by 0.003 μM thioesterase), and transient-state multiple-turnover reaction (50 μM [(14)C]-4-HB-CoA catalyzed by 10 μM thioesterase). Together with the results obtained from solvent (18)O labeling experiments, the findings are interpreted as evidence of the formation of an aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate that undergoes rate-limiting hydrolytic cleavage at the hydroxybenzoyl carbonyl carbon atom.     

Reductive, coenzyme A-mediated pathway for 3-chlorobenzoate degradation in the phototrophic bacterium Rhodopseudomonas palustris

We isolated a strain of Rhodopseudomonas palustris (RCB100) by selective enrichment in light on 3-chlorobenzoate to investigate the steps that it uses to accomplish anaerobic dechlorination. Analyses of metabolite pools as well as enzyme assays suggest that R. palustris grows on 3-chlorobenzoate by (i) converting it to 3-chlorobenzoyl coenzyme A (3-chlorobenzoyl-CoA), (ii) reductively dehalogenating 3-chlorobenzoyl-CoA to benzoyl-CoA, and (iii) degrading benzoyl-CoA to acetyl-CoA and carbon dioxide. R. palustris uses 3-chlorobenzoate only as a carbon source and thus incorporates the acetyl-CoA that is produced into cell material. The reductive dechlorination route used by R. palustris for 3-chlorobenzoate degradation differs from those previously described in that a CoA thioester, rather than an unmodified aromatic acid, is the substrate for complete dehalogenation.     

Cloning and sequence analysis of genes for dehalogenation of 4-chlorobenzoate from Arthrobacter sp. strain SU.

Strains of Arthrobacter catalyze a hydrolytic dehalogenation of 4-chlorobenzoate (4-CBA) to p-hydroxybenzoate. The reaction requires ATP and coenzyme A (CoA), indicating activation of the substrate via a thioester, like that reported for Pseudomonas sp. strain CBS3 (J. D. Scholten, K.-H. Chang, P. C. Babbit, H. Charest, M. Sylvestre, and D. Dunaway-Mariano, Science 253:182-185, 1991). The dehalogenase genes of Arthrobacter sp. strain SU were cloned and expressed in Escherichia coli. Analyses of deletions indicate that dehalogenation depends on three open reading frames (ORFs) which are organized in an operon. There is extensive sequence homology to corresponding gene products in Pseudomonas sp. strain CBS3, suggesting that ORF1 and ORF2 encode a 4-CBA-CoA-ligase and a 4-CBA-CoA dehalogenase, respectively. ORF3 possibly represents a thioesterase, although no homology to the enzyme from Pseudomonas sp. strain CBS3 exists.     

Cloning and sequence analysis of genes for dehalogenation of 4-chlorobenzoate from Arthrobacter sp. strain SU.

Strains of Arthrobacter catalyze a hydrolytic dehalogenation of 4-chlorobenzoate (4-CBA) to p-hydroxybenzoate. The reaction requires ATP and coenzyme A (CoA), indicating activation of the substrate via a thioester, like that reported for Pseudomonas sp. strain CBS3 (J. D. Scholten, K.-H. Chang, P. C. Babbit, H. Charest, M. Sylvestre, and D. Dunaway-Mariano, Science 253:182-185, 1991). The dehalogenase genes of Arthrobacter sp. strain SU were cloned and expressed in Escherichia coli. Analyses of deletions indicate that dehalogenation depends on three open reading frames (ORFs) which are organized in an operon. There is extensive sequence homology to corresponding gene products in Pseudomonas sp. strain CBS3, suggesting that ORF1 and ORF2 encode a 4-CBA-CoA-ligase and a 4-CBA-CoA dehalogenase, respectively. ORF3 possibly represents a thioesterase, although no homology to the enzyme from Pseudomonas sp. strain CBS3 exists.     

Dehalogenation of 4-chlorobenzoate by 4-chlorobenzoate dehalogenase from pseudomonas sp. CBS3: an ATP/coenzyme A dependent reaction

Pseudomonas sp. CBS3 was grown with 4-chlorobenzoate as sole source of carbon and energy. Freshly prepared cell-free extracts converted 4-chlorobenzoate to 4-hydroxybenzoate. After storage for 16 hours at 25 degrees C only about 50% of the initial activity was left. Treatment at 55 degrees C for 10 minutes, dialysis or desalting of the extracts by gel filtration caused a total loss of the activity of the 4-chlorobenzoate dehalogenase. The activity could be restored by the addition of ATP, coenzyme A and Mg2+. If one of these cofactors was missing, no dehalogenating activity was detectable. The amount of 4-hydroxybenzoate formed was proportional to the amount of ATP available in the test system whereas CoA served as a real coenzyme. A novel ATP/coenzyme A dependent reaction mechanism for the dehalogenation of 4-chlorobenzoate by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 is proposed.     

Characterization of the 4-Hydroxybenzoyl-Coenzyme A Thioesterase from Arthrobacter sp. Strain SU

The Arthrobacter sp. strain SU 4-chlorobenzoate (4-CBA) dehalogenation pathway converts 4-CBA to 4-hydroxybenzoate (4-HBA). The pathway operon contains the genes fcbA, fcbB, and fcbC (A. Schmitz, K. H. Gartemann, J. Fiedler, E. Grund, and R. Eichenlaub, Appl. Environ. Microbiol. 58:4068-4071, 1992). Genes fcbA and fcbB encode 4-CBA-coenzyme A (CoA) ligase and 4-CBA-CoA dehalogenase, respectively, whereas the function of fcbC is not known. We subcloned fcbC and expressed it in Escherichia coli, and we purified and characterized the FcbC protein. A substrate activity screen identified benzoyl-CoA thioesters as the most active substrates. Catalysis of 4-HBA-CoA hydrolysis to 4-HBA and CoA occurred with a k_cat of 6.7 s⁻¹ and a K_m of 1.2 μM. The k_cat pH rate profile for 4-HBA-CoA hydrolysis indicated optimal activity over a pH range of 6 to 10. The amino acid sequence of the FcbC protein was compared to other sequences contained in the protein sequence data banks. A large number of sequence homologues of unknown function were identified. On the other hand, the 4-HBA-CoA thioesterases isolated from 4-CBA-degrading Pseudomonas strains did not share significant sequence identity with the FcbC protein, indicating early divergence of the thioesterase-encoding genes.     

Structure and denaturation of 4-chlorobenzoyl coenzyme A dehalogenase from <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. strain TM-1

The secondary structure of the trimeric protein 4-chlorobenzoyl coenzyme A dehalogenase from Arthrobacter sp. strain TM-1, the second of three enzymes involved in the dechlorination of 4-chlorobenzoate to form 4-hydroxybenzoate, has been examined. E_mM for the enzyme was 12.59. Analysis by circular dichroism spectrometry in the far uv indicated that 4-chlorobenzoyl coenzyme A dehalogenase was composed mostly of α-helix (56%) with lesser amounts of random coil (21%), β-turn (13%) and β-sheet (9%). These data are in close agreement with a computational prediction of secondary structure from the primary amino acid sequence, which indicated 55.8% α-helix, 33.7% random coil and 10.5% β-sheet; the enzyme is, therefore, similar to the 4-chlorobenzoyl coenzyme A dehalogenase from Pseudomonas sp. CBS-3. The three-dimensional structure, including that of the presumed active site, predicted by computational analysis, is also closely similar to that of the Pseudomonas dehalogenase. Study of the stability and physicochemical properties revealed that at room temperature, the enzyme was stable for 24 h but was completely inactivated by heating to 60°C for 5 min; thereafter by cooling at 1°C min⁻¹ to 45°C, 20.6% of the activity could be recovered. Mildly acidic (pH 5.2) or alkaline (pH 10.1) conditions caused complete inactivation, but activity was fully recovered on returning the enzyme to pH 7.4. Circular dichroism studies also indicated that secondary structure was little altered by heating to 60°C, or by changing the pH from 7.4 to 6.0 or 9.2. Complete, irreversible destruction of, and maximal decrease in the fluorescence yield of the protein at 330–350 nm were brought about by 4.5 M urea or 1.1 M guanidinium chloride. Evidence was obtained to support the hypothetical three-dimensional model, that residues W140 and W167 are buried in a non-polar environment, whereas W182 appears at or close to the surface of the protein. At least one of the enzymes of the dehalogenase system (the combined 4-chlorobenzoate:CoA ligase, the dehalogenase and 4-hydroxybenzoyl coenzyme A thioesterase) appears to be capable of association with the cell membrane.

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.     

Metabolism of l-β-lysine by a Pseudomonas: Purification and properties of 3-keto-6-acetamidohexanoate cleavage enzyme

Extracts of Pseudomonas B4 grown with l-β-lysine (3,6-diaminohexanoate) as the main energy source are shown to contain a 3-keto-6-acetamidohexanoate cleavage enzyme that converts 3-keto-6-acetamidohexanoate and acetyl · CoA reversibly to 4-acetamidobutyryl · CoA and acetoacetate. The enzyme catalyzes the third step in β-lysine degradation. In unfractionated extracts cleavage enzyme activity is generally assayed spectrophotometrically by coupling the forward reaction with excess 4-acetamidobutyryl · CoA thiolesterase, derived from the same organism, and measuring the rate of CoASH formation by reaction with 5,5-dithiobis(2-nitrobenzoic acid). Enzyme freed of thiolesterase is conveniently assayed by using 4-acetamidobutyryl · CoA and acetoacetate as substrates and measuring acetyl · CoA formation by means of citrate synthase reaction in the presence of 5,5-dithiobis(2-nitrobenzoic acid). The cleavage enzyme has been purified 38-fold to a specific activity of 237 mU/mg. The stoichiometry, equilibrium constant, molecular weight, and various kinetic properties of the enzymatic reaction have been determined. The substrate specificity of the Pseudomonas enzyme differs markedly from that of the analogous 3-keto-5-aminohexanoate cleavage enzyme of Clostridium subterminale strain SB4 and is broader. In the forward reaction 3-ketohexanoate can replace 3-keto-6-acetamidohexanoate, and propionyl · CoA can replace acetyl · CoA as a substrate. In the backward reaction, 4-acetamidobutyryl · CoA can be replaced by any of several CoA thiolesters including the butyryl, valeryl, 4-propionamidobutyryl, 3-acetamidopropionyl, and β-alanyl derivatives, and acetoacetate can be replaced by 2-methylacetoacetate. The products of these reactions have been characterized. Unlike the cleavage enzyme of Clostridium subterminale strain SB4, the Pseudomonas enzyme is not stimulated by Co²⁺ or Mn²⁺ and is not inhibited by EDTA, 5,5-dithiobis(2-nitrobenzoic acid), or p-chloromercuribenzoate. Tracer experiments indicate that carbon atoms 1 and 2 of acetoacetate are derived from carbon atoms 1 and 2 of 3-keto-6-acetamidohexanoate, and carbon atoms 3 and 4 of acetoacetate are derived from the acetyl group of acetyl · CoA. The cleavage enzyme is not formed in detectable amounts when Pseudomonas B4 is grown in a peptone-yeast extract medium.     

Synthesis and antimicrobial activity of novel fluorine containing 4-(substituted-2-hydroxybenzoyl)-1H-pyrazoles and pyrazolyl benzo[d]oxazoles

A series of fluorine containing 4-(substituted-2-hydroxybenzoyl) pyrazoles and pyrazolyl benzo[d]oxazoles were synthesized and evaluated for their antibacterial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis and antifungal activity against Candida albicans. The antibacterial activities were expressed as the minimum inhibitory concentration (MIC₅₀) in μg/ml. The compounds 1-(3,4-difluorophenyl)-4-(5-fluoro-2-hydroxybenzoyl)-1H-pyrazole (4b), oxime derivatives such as 1-(3,4-difluorophenyl)-1H-pyrazol-4-yl)(2-hydroxy-4-methylphenyl)methanone oxime (5b) and (5-chloro-2-hydroxyphenyl)(1-(3,4-difluorophenyl)-1H-pyrazol-4-yl)methanone oxime (5e) exhibited promising activities against tested bacterial strains. Except compound 1-(3,4-difluorophenyl)-4-(2-hydroxybenzoyl)-1H-pyrazole (4d), none of the other compounds showed promising antifungal activity.     

Formation of 3-hexaprenyl-4-hydroxybenzoate by matrix-free mitochondrial membrane-rich preparations of yeast.

It has been shown that a 10 000 x g matrix-free mitochondrial membrane-rich preparation from commercial bakers' yeast is able to synthesize 3-all-transhexaprenyl-4-hydroxybenzoate from 4-hydroxybenzoate and isopentenyl pyrophosphate. The synthesis is Mg2+ dependent and is stimulated markedly by the primer for polyprenylpyrophosphate synthesis of 3-hexaprenyl-4-hydroxybenzoate from 4-hydroxybenzoate, isopentenyl pyrophosphate and 3,3-dimethylallyl pyrophosphate the priming function of 3,3-dimethylallyl pyrophosphate can be performed by either geranyl pyrophosphate (most efficient) or farnesyl pyrophosphate. At high Mg2+ concentrations, however, geranyl pyrophosphate and farnesyl pyrophosphate act mainly as sources of preformed side chains and 3-diprenyl- and 3-tripenyl-4-hydroxybenzoate, respectively, are produced. In the presence of a source of preformed polyprenyl pyrophosphates the membrane preparations catalysed the polyprenylation of methyl-4-hydroxybenzoate, 4-hydroxybenzaldehyde, 4-hydroxybenzylalcohol and 4-hydroxycinnamate. No evidence was obtained for the involvement of either 4-hydroxybenzoyl CoA or 4-hydroxybenzoyl-S-protein in the formation of 3-polyprenyl-4-hydroxybenzoates.     

Enzymes involved in the anaerobic degradation of meta-substituted halobenzoates

Organohalides are environmentally relevant compounds that can be degraded by aerobic and anaerobic microorganisms. The denitrifying Thauera chlorobenzoica is capable of degrading halobenzoates as sole carbon and energy source under anaerobic conditions. LC‐MS/MS‐based coenzyme A (CoA) thioester analysis revealed that 3‐chloro‐ or 3‐bromobenzoate were preferentially metabolized via non‐halogenated CoA‐ester intermediates of the benzoyl‐CoA degradation pathway. In contrast, 3‐fluorobenzoate, which does not support growth, was converted to dearomatized fluorinated CoA ester dead‐end products. Extracts from cells grown on 3‐chloro‐/3‐bromobenzoate catalysed the Ti(III)‐citrate‐ and ATP‐dependent reductive dehalogenation of 3‐chloro/3‐bromobenzoyl‐CoA to benzoyl‐CoA, whereas 3‐fluorobenzoyl‐CoA was converted to a fluorinated cyclic dienoyl‐CoA compound. The reductive dehalogenation reactions were identified as previously unknown activities of ATP‐dependent class I benzoyl‐CoA reductases (BCR) present in all facultatively anaerobic, aromatic compound degrading bacteria. A two‐step dearomatization/H‐halide elimination mechanism is proposed. A halobenzoate‐specific carboxylic acid CoA ligase was characterized in T. chlorobenzoica; however, no such enzyme is present in Thauera aromatica, which cannot grow on halobenzoates. In conclusion, it appears that the presence of a halobenzoate‐specific carboxylic acid CoA ligase rather than a specific reductive dehalogenase governs whether an aromatic compound degrading anaerobe is capable of metabolizing halobenzoates.     

A novel 4-hydroxycoumarin biosynthetic pathway

Coumarin forms in melilotoside (trans-ortho-coumaric acid glucoside)-containing plant species upon cell damage. In moldy melilotoside-containing plant material, trans-ortho-coumaric acid is converted by fungi to 4-hydroxycoumarin, two molecules of which spontaneously combine with formaldehyde to give dicoumarol. Dicoumarol causes internal bleeding in livestock and is the forerunner of the warfarin group of medicinal anticoagulants. Here, we report 4-hydroxycoumarin formation by biphenyl synthase (BIS). Two new BIS cDNAs were isolated from elicitor-treated Sorbus aucuparia cell cultures. The encoded isoenzymes preferred ortho-hydroxybenzoyl (salicoyl)-CoA as a starter substrate and catalyzed a single decarboxylative condensation with malonyl-CoA to give 4-hydroxycoumarin. When elicitor-treated S. aucuparia cell cultures were fed with the N-acetylcysteamine thioester of salicylic acid, 4-hydroxycoumarin accumulated in the culture medium. Incubation of the BIS isoenzymes with benzoyl-CoA and malonyl-CoA resulted in the formation of 3,5-dihydroxybiphenyl which is the precursor of the phytoalexins of the Maloideae.     

A Novel Hydrolytic Dehalogenase for the Chlorinated Aromatic Compound Chlorothalonil

Dehalogenases play key roles in the detoxification of halogenated aromatics. Interestingly, only one hydrolytic dehalogenase for halogenated aromatics, 4-chlorobenzoyl-coenzyme A (CoA) dehalogenase, has been reported. Here, we characterize another novel hydrolytic dehalogenase for a halogenated aromatic compound from the 2,4,5,6-tetrachloroisophthalonitrile (chlorothalonil)-degrading strain of Pseudomonas sp. CTN-3, which we have named Chd. Chd catalyzes a hydroxyl substitution at the 4-chlorine atom of chlorothalonil. The metabolite of the Chd dehalogenation, 4-hydroxy-trichloroisophthalonitrile, was identified by reverse-phase high-performance liquid chromatography (HPLC), tandem mass spectrometry (MS/MS), and nuclear magnetic resonance (NMR). Chd dehalogenates chlorothalonil under anaerobic and aerobic conditions and does not require the presence of cofactors such as CoA and ATP. Chd contains a putative conserved domain of the metallo-β-lactamase superfamily and shows the highest identity with several metallohydrolases (24 to 29%). Chd is a monomer (36 kDa), and the isoelectric point (pI) of Chd is estimated to be 4.13. Chd has a dissociation constant (K_m) of 0.112 mM and an overall catalytic rate (k_cat) of 207 s⁻¹ for chlorothalonil. Chd is completely inhibited by 1,10-phenanthroline, diethyl pyrocarbonate, and N-bromosuccinic acid. Site-directed mutagenesis of Chd revealed that histidines 128 and 157, serine 126, aspartates 45, 130 and 184, and tryptophan 241 were essential for the dehalogenase activity. Chd differs from other reported hydrolytic dehalogenases based on the analysis of amino acid sequences and catalytic mechanisms. This study provides an excellent dehalogenase candidate for mechanistic study of hydrolytic dehalogenation of halogenated aromatic compound.Halogenated aromatic compounds are widely used in agriculture and industry as solvents, defatting agents, herbicides, and fungicides. A variety of these compounds have been identified as priority organic pollutants by the United Nations and the U.S. Environmental Protection Agency. Therefore, the remediation of these pollutants is desirable. Microorganisms play key roles in the detoxification of halogenated aromatics. The use of microbial enzymes for bioremediation has received increasing attention as an efficient and cost-effective biotechnological approach. Halogen removal from halogenated aromatics reduces both the recalcitrance to biodegradation and the risk of forming toxic intermediates during subsequent metabolic steps. As a result, the key reaction for microbial detoxification of halogenated aromatics is the actual dehalogenation (35).Investigation of the microbial degradation of different halogenated aromatics has led to the detection and elucidation of various dehalogenases that catalyze the removal of the halogen atom under aerobic and anaerobic conditions (8, 11, 17). Four dehalogenation mechanisms of halogenated aromatics are known, including reductive, thiolytic, oxidative, and hydrolytic mechanisms (41). Reductive dehalogenation plays important roles in the degradation of chlorinated aromatics under anaerobic conditions (36, 44). Several anaerobic bacteria are capable of using chlorinated benzenes (2) or polychlorinated dibenzodioxins (5) as the terminal electron acceptors in their energy metabolism. These bacteria couple reductive dehalogenation to electron transport phosphorylation (15). Several enzymes catalyzing the respiratory reductive dechlorination of halogenated aromatics have also been characterized (1, 4, 7, 20, 28, 38, 40). Under aerobic conditions, some chlorinated aromatics can also be reductively dehalogenated by thiolytic substitution in the presence of glutathione (12, 19, 25, 46, 48). In this dehalogenation system, chlorine atoms are displaced by the nucleophilic attack of the thiolate anion of glutathione. The nucleophilic attack of the thiolate anion is catalyzed by glutathione S-transferases (43). Besides the two well-characterized mechanisms for aryl halide reductive dehalogenation, two other mechanisms have been reported, including reduced NADPH-dependent reductive dechlorination of 2,4-dichlorobenzoyl-coenzyme A (CoA) to 4-chlorobenzoyl-CoA in Corynebacterium sepedonicum KZ-4 and coryneform bacterium strain NTB-1 (31), as well as a CoA-mediated reductive dehalogenation of 3-chlorobenzoate in Rhodopseudomonas palustris RCB100 using 3-chlorobenzoate as the carbon source rather than as a terminal electron acceptor (13). Oxidative dehalogenation of halogenated aromatics is catalyzed by monooxygenase (29), dioxygenases (34, 37, 39, 45), and peroxidase (30).Even though several hydrolytic dehalogenases involved in dehalogenation of halogenated aliphatic hydrocarbons and halogenated carboxylic acids have been characterized, only one kind of hydrolytic dehalogenase for halogenated aromatics has been reported. The only hydrolytic dehalogenase identified to date is 4-chlorobenzoyl-CoA dehalogenase in the 4-chlorobenzoate degradation system (32, 33). For the hydrolytic substitution of the chlorine atom of 4-chlorobenzoate with a hydroxyl group, activation by the CoA thioester formation is required. Initially, 4-chlorobenzoate-CoA ligase adenylates the carboxyl group in a reaction requiring ATP, followed by the replacement of AMP with CoA and the formation of a thioester. This intermediate is sufficiently energized to facilitate the replacement of the hydroxyl group with a 4-chlorine atom; this is catalyzed by 4-chlorobenzoyl-CoA dehalogenase. Finally, 4-hydroxybenzoyl-CoA thioesterase removes the CoA (Fig. ​(Fig.1a).1a). Three separate enzymes are involved in this system, and cofactors including CoA and ATP are needed (32, 33).Open in a separate windowFIG. 1.Dechlorination mechanism of 4-chlorobenzoate in Pseudomonas sp. CBS3 and Acinetobacter sp. strain 4-CB1 (a) and the first-step dechlorination mechanism of 2,4,5,6-tetrachloroisophthalonitrile (chlorothalonil) in Pseudomonas sp. CTN-3 (b).Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile), a broad-spectrum chlorinated aromatic fungicide, is the second most widely used agricultural fungicide in the United States, with 5 million kilograms applied annually (9). Chlorothalonil is highly toxic to fish, birds, and aquatic invertebrates (6) and is commonly detected in ecosystems (18). The bacterial strain Pseudomonas sp. CTN-3, capable of efficiently transforming chlorothalonil, was isolated in our laboratory from long-term chlorothalonil-contaminated soil in the Jiangsu Province in China. In Ochrobactrum anthropi SH35B, a glutathione-dependent glutathione S-transferase was reported to be able to catalyze the nucleophilic substitution of chlorine atoms of chlorothalonil (19). The glutathione S-transferase in our CTN-3 bacterial strain showed 84% identity with that of O. anthropi SH35B. However, the glutathione S-transferase from CTN-3 was not functionally expressed. Here, we report for the first time the characterization of a novel chlorothalonil hydrolytic dehalogenase (Chd) that contains a conserved domain of the metallo-β-lactamase superfamily. Chd is the second hydrolytic dehalogenase for chlorinated aromatic compounds to be identified. The hydrolytic dehalogenation of chlorothalonil catalyzed by Chd is independent of CoA and ATP. Based on the analysis of amino acid sequences and catalytic mechanisms, Chd is unique from the other reported hydrolytic dehalogenases.     

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.