Biodegradation and metabolic pathway of β-chlorinated aliphatic acid in Bacillus sp. CGMCC no. 4196

In this study, a bacterial Bacillus sp. CGMCC no. 4196 was isolated from mud. This strain exhibited the ability to degrade high concentration of 3-chloropropionate (3-CPA, 120 mM) or 3-chlorobutyrate (30 mM), but not chloroacetate or 2-chloropropionate (2-CPA). The growing cells, resting cells, and cell-free extracts from this bacterium had the capability of 3-CPA degradation. The results indicated that the optimum biocatalyst for 3-CPA biodegradation was the resting cells. The 3-CPA biodegradation pathway was further studied through the metabolites and critical enzymes analysis by HPLC, LC-MS, and colorimetric method. The results demonstrated that the metabolites of 3-CPA were 3-hydroxypropionic acid (3-HP) and malonic acid semialdehyde, and the critical enzymes were 3-CPA dehalogenase and 3-HP dehydroxygenase. Thus, the mechanism of the dehalogenase-catalyzed reaction was inferred as hydrolytic dehalogenation which was coenzyme A-independent and oxygen-independent. Finally, the pathway of β-chlorinated aliphatic acid biodegradation could be concluded as follows: the β-chlorinated acid is first hydrolytically dehalogenated to the β-hydroxyl aliphatic acid, and the hydroxyl aliphatic acid is oxidized to β-carbonyl aliphatic acid by β-hydroxy aliphatic acid dehydroxygenase. It is the first report that 3-HP was produced from 3-CPA by β-chlorinated aliphatic acid dehalogenase.     

Purification and properties of a new enzyme, DL-2-haloacid dehalogenase, from Pseudomonas sp.

A new enzyme, DL-2-haloacid dehalogenase, was isolated and purified to homogeneity from the cells of Pseudomonas sp. strain 113. This enzyme catalyzed non-stereospecific dehalogenation of both of the optical isomers of 2-chloropropionate through an SN2 type of reaction; L- and D-lactates were formed from D- and L-2-chloropropionates, respectively. The enzyme acted on 2-halogenated aliphatic carboxylic acids whose carbon chain lengths were less than five. It also dehalogenated trichloroacetate to form oxalate and showed maximum activity at pH 9.5. The Michaelis constants for substrates were as follows: 5.0 mM for monochloroacetate, 1.1 mM for L-2-chloropropionate, and 4.8 mM for D-2-chloropropionate. DL-2-Haloacid dehalogenase was inhibited by HgCl2, ZnSO4, and MnSO4, but was not affected by thiol reagents, such as p-chloromercuribenzoate and iodoacetamide. This enzyme had a molecular weight of about 68,000 and appeared to be composed of two subunits identical in molecular weight.     

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.     

Aliphatic and chlorinated alkenes and epoxides as inducers of alkene monooxygenase and epoxidase activities in Xanthobacter strain Py2.

The inducible nature of the alkene oxidation system of Xanthobacter strain Py2 has been investigated. Cultures grown with glucose as the carbon source did not contain detectable levels of alkene monooxygenase or epoxidase, two key enzymes of alkene and epoxide metabolism. Upon addition of propylene to glucose-grown cultures, alkene monooxygenase and epoxidase activities increased and after an 11-h induction period reached levels of specific activity comparable to those in propylene-grown cells. Addition of chloramphenicol or rifampin prevented the increase in the enzyme activities. Comparison of the banding patterns of proteins present in cell extracts revealed that polypeptides with molecular masses of 43, 53, and 57 kDa accumulate in propylene-grown but not glucose-grown cells. Pulse-labeling of glucose-grown cells with [35S]methionine and [35S]cysteine revealed that the 43-, 53-, and 57-kDa proteins, as well as two additional polypeptides with molecular masses of 12 and 21 kDa, were newly synthesized upon exposure of cells to propylene or propylene oxide. The addition to glucose-grown cells of a variety of other aliphatic and chlorinated alkenes and epoxides, including ethylene, vinyl chloride (1-chloroethylene), cis- and trans-1,2-dichloroethylene, 1-chloropropylene, 1,3-dichloropropylene, 1-butylene, trans-2-butylene, isobutylene, ethylene oxide, epichlorohydrin (3-chloro-1,2-epoxypropane), 1,2-epoxybutane, cis- and trans-2,3-epoxybutane, and isobutylene oxide stimulated the synthesis of the five propylene-inducible polypeptides as well as increases in alkene monooxygenase and epoxidase activities. In contrast, acetylene, and a range of aliphatic and chlorinated alkanes, did not stimulate the synthesis of the propylene-inducible polypeptides or the increase in alkene monooxygenase and epoxidase activities.     

Bacterial DL-2-haloacid dehalogenase from Pseudomonas sp. strain 113: gene cloning and structural comparison with D- and L-2-haloacid dehalogenases.

DL-2-Haloacid dehalogenase from Pseudomonas sp. strain 113 (DL-DEX) catalyzes the hydrolytic dehalogenation of both D- and L-2-haloalkanoic acids to produce the corresponding L- and D-2-hydroxyalkanoic acids, respectively, with inversion of the C2 configuration. DL-DEX is a unique enzyme: it acts on the chiral carbon of the substrate and uses both enantiomers as equivalent substrates. We have isolated and sequenced the gene encoding DL-DEX. The open reading frame consists of 921 bp corresponding to 307 amino acid residues. No sequence similarity between DL-DEX and L-2-haloacid dehalogenases was found. However, DL-DEX had significant sequence similarity with D-2-haloacid dehalogenase from Pseudomonas putida AJ1, which specifically acts on D-2-haloalkanoic acids: 23% of the total amino acid residues of DL-DEX are conserved. We mutated each of the 26 residues with charged and polar side chains, which are conserved between DL-DEX and D-2-haloacid dehalogenase. Thr65, Glu69, and Asp194 were found to be essential for dehalogenation of not only the D- but also the L-enantiomer of 2-haloalkanoic acids. Each of the mutant enzymes, whose activities were lower than that of the wild-type enzyme, acted on both enantiomers of 2-haloacids as equivalent substrates in the same manner as the wild-type enzyme. We also found that each enantiomer of 2-chloropropionate competitively inhibits the enzymatic dehalogenation of the other. These results suggest that DL-DEX has a single and common catalytic site for both enantiomers.     

A new -2-haloacid dehalogenase acting on 2-haloacid amides: purification, characterization, and mechanism

-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of - and -2-haloalkanoic acids to produce the corresponding - and -2-hydroxyalkanoic acids, respectively. We have constructed an overproduction system for -2-haloacid dehalogenase from Pseudomonas putida PP3 ( -DEX 312) and purified the enzyme to analyze the reaction mechanism. When a single turnover reaction of -DEX 312 was carried out in H₂¹⁸O by use of a large excess of the enzyme with - or -2-chloropropionate as a substrate, the lactate produced was labeled with ¹⁸O. This indicates that the solvent water molecule directly attacked the substrate and that its oxygen atom was incorporated into the product. This reaction mechanism contrasts with that of -2-haloacid dehalogenase, which has an active-site carboxylate group that attacks the substrate to displace the halogen atom. -DEX 312 resembles -2-haloacid dehalogenase from Pseudomonas sp. 113 ( -DEX 113) in that the reaction proceeds with a direct attack of a water molecule on the substrate. However, -DEX 312 is markedly different from -DEX 113 in its substrate specificity. We found that -DEX 312 catalyzes the hydrolytic dehalogenation of 2-chloropropionamide and 2-bromopropionamide, which do not serve as substrates for -DEX 113. -DEX 312 is the first enzyme that catalyzes the dehalogenation of 2-haloacid amides.     

Purification and characterization of thermostable and nonthermostable 2-haloacid dehalogenases with different stereospecificities from Pseudomonas sp. strain YL.

Two novel hydrolytic dehalogenases, thermostable L-2-haloacid dehalogenase (L-DEX) inducibly synthesized by 2-chloropropionate (2-CPA) and nonthermostable DL-2-haloacid dehalogenase (DL-DEX) induced by 2-chloroacrylate, were purified to homogeneity from Pseudomonas sp. strain YL. DL-DEX consisted of a monomer with a molecular weight of about 36,000 and catalyzed the dehalogenation of L and D isomers of 2-CPA to produce D- and L-lactates, respectively. It acted on 2-haloalkanoic acids with a carbon chain length of 2 to 4. The maximum activity on DL-2-CPA was found at pH 10.5 and 45 degrees C. L-DEX, composed of two subunits with identical molecular weights of 27,000, catalyzes the dehalogenation of L-2-haloalkanoic acids to produce the corresponding D-2-hydroxyalkanoic acids. The enzyme acts not only on short-carbon-chain 2-haloacids such as monochloroacetate and monoiodoacetate in aqueous solution but also on long-carbon-chain 2-haloacids such as 2-bromohexadecanoate in n-heptane. L-DEX is thermostable: it retained its full activity upon heating at 60 degrees C for 30 min. The pH and temperature optima for dehalogenation of L-2-CPA were 9.5 and 65 degrees C, respectively. L-DEX was strongly inhibited by modification of carboxyl groups with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and Woodward reagent K, but DL-DEX was not.     

Fermentation of 1,2-Propanediol and 1,2-Ethanediol by Some Genera of Enterobacteriaceae, Involving Coenzyme B12-Dependent Diol Dehydratase

Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 was able to grow anaerobically on 1,2-propanediol and 1,2-ethanediol as carbon and energy sources. Whole cells of the bacterium grown anaerobically on 1,2-propanediol or on glycerol catalyzed conversion of 1,2-diols and aldehydes to the corresponding acids and alcohols. Glucose-grown cells also converted aldehydes, but not 1,2-diols, to acids and alcohols. The presence of activities of coenzyme B(12)-dependent diol dehydratase, alcohol dehydrogenase, coenzyme-A-dependent aldehyde dehydrogenase, phosphotransacetylase, and acetate kinase was demonstrated with crude extracts of 1,2-propanediol-grown cells. The dependence of the levels of these enzymes on growth substrates, together with cofactor requirements in in vitro conversion of these substrates, indicates that 1,2-diols are fermented to the corresponding acids and alcohols via aldehydes, acyl-coenzyme A, and acyl phosphates. This metabolic pathway for 1,2-diol fermentation was also suggested in some other genera of Enterobacteriaceae which were able to grow anaerobically on 1,2-propanediol. When the bacteria were cultivated in a 1,2-propanediol medium not supplemented with cobalt ion, the coenzyme B(12)-dependent conversion of 1,2-diols to aldehydes was the rate-limiting step in this fermentation. This was because the intracellular concentration of coenzyme B(12) was very low in the cells grown in cobalt-deficient medium, since the apoprotein of diol dehydratase was markedly induced in the cells grown in the 1,2-propanediol medium. Better cell yields were obtained when the bacteria were grown anaerobically on 1,2-propanediol. Evidence is presented that aerobically grown cells have a different metabolic pathway for utilizing 1,2-propanediol.     

Cometabolism of 3,4-dichlorobenzoate by Acinetobacter sp. strain 4-CB1.

When Acinetobacter sp. strain 4-CB1 was grown on 4-chlorobenzoate (4-CB), it cometabolized 3,4-dichlorobenzoate (3,4-DCB) to 3-chloro-4-hydroxybenzoate (3-C-4-OHB), which could be used as a growth substrate. No cometabolism of 3,4-DCB was observed when Acinetobacter sp. strain 4-CB1 was grown on benzoate. 4-Carboxyl-1,2-benzoquinone was formed as an intermediate from 3,4-DCB and 3-C-4-OHB in aerobic and anaerobic resting-cell incubations and was the major transient intermediate found when cells were grown on 3-C-4-OHB. The first dechlorination step of 3,4-DCB was catalyzed by the 4-CB dehalogenase, while a soluble dehalogenase was responsible for dechlorination of 3-C-4-OHB. Both enzymes were inducible by the respective chlorinated substrates, as indicated by oxygen uptake experiments. The dehalogenase activity on 3-C-4-OHB, observed in crude cell extracts, was 109 and 44 nmol of 3-C-4-OHB min-1 mg of protein-1 under anaerobic and aerobic conditions, respectively. 3-Chloro-4-hydroxybenzoate served as a pseudosubstrate for the 4-hydroxybenzoate monooxygenase by effecting oxygen and NADH consumption without being hydroxylated. Contrary to 4-CB metabolism, the results suggest that 3-C-4-OHB was not metabolized via the protocatechuate pathway. Despite the ability of resting cells grown on 4-CB or 3-C-4-OHB to carry out all of the necessary steps for dehalogenation and catabolism of 3,4-DCB, it appeared that 3,4-DCB was unable to induce the necessary 4-CB dehalogenase for the initial p-dehalogenation step.(ABSTRACT TRUNCATED AT 250 WORDS)     

beta-Ketoadipate pathway in Trichosporon cutaneum modified for methyl-substituted metabolites.

Trichosporon cutaneum, when grown with p-cresol, catalyzed intradiol fission of the benzene nucleus of 4-methylcatechol before the complete catabolism of these two substrates. Steps in their conversion to pyruvate and acetyl coenzyme A were investigated by using cell extracts, and some properties of various new microbial catabolites are also described. These included (-)-2,5-dihydro-3-methyl-5-oxofuran-2-acetic acid (beta-methylmuconolactone) and (-)-3-keto-4-methyladipic acid and its coenzyme A ester; the latter was degraded by an enzymatic reaction sequence that included the coenzyme A esters of methylsuccinic, itaconic, and citramalic acids. A notable feature of this sequence is the formation of beta-methylmuconolactone which can be readily metabolized, in contrast to the analogous reaction in bacteria that gives the "dead-end" compound gamma-methylmuconolactone; this compound cannot be enzymatically degraded and so renders the beta-ketoadipate pathway unavailable for methyl-substituted bacterial sources of carbon that are catabolized by way of 4-methylcatechol.     

A mechanistic analysis of enzymatic degradation of organohalogen compounds

Enzymes that catalyze the conversion of organohalogen compounds have been attracting a great deal of attention, partly because of their possible applications in environmental technology and the chemical industry. We have studied the mechanisms of enzymatic degradation of various organic halo acids. In the reaction of L-2-haloacid dehalogenase and fluoroacetate dehalogenase, the carboxylate group of the catalytic aspartate residue nucleophilically attacked the α-carbon atom of the substrates to displace the halogen atom. In the reaction catalyzed by DL-2-haloacid dehalogenase, a water molecule directly attacked the substrate to displace the halogen atom. In the course of studies on the metabolism of 2-chloroacrylate, we discovered two new enzymes. 2-Haloacrylate reductase catalyzed the asymmetric reduction of 2-haloacrylate to produce L-2-haloalkanoic acid in an NADPH-dependent manner. 2-Haloacrylate hydratase catalyzed the hydration of 2-haloacrylate to produce pyruvate. The enzyme is unique in that it catalyzes the non-redox reaction in an FADH(2)-dependent manner.     

A comparative study of acquired amidase activity in Pseudomonas species

Pseudomonas putida PP3 carrying dehalogenases I and II and Pseudomonas aeruginosa PAU3 carrying dehalogenase I coded for by plasmid pUU2 were able to grow on 2-monochloropropionic acid (2MCPA). Neither strain utilized 2-chloropropionamide (2CPA) as a carbon or nitrogen source for growth. Mutations in both strains to 2Cpa+ phenotypes (designated P. putida PPW3 and P. aeruginosa PAU5, respectively) involved the expression of an acquired 2CPA-amidase activity. The amidase followed by dehalogenase reactions in these strains constituted a novel metabolic pathway for growth on 2CPA. P. putida PPW3 synthesized a constitutive amidase of molecular mass 59 kDa consisting of two identical subunits of 29 kDa. For those amides tested this acquired enzyme was most active against chlorinated aliphatic amides, although substrate affinities (Km) and maximum rates of activity (Vmax) were poor. P. aeruginosa PAU5 acquired a 2Cpa+ phenotype by overproducing the A-amidase normally used by this species to hydrolyse aliphatic amides. The A-amidase had only slight activity towards 2CPA. However, with constitutive synthesis the mutant grew on the chlorinated substrates. Chloroacetamide (CAA) was a toxic substrate analogue for these Pseudomonas strains. A strain resistant to CAA was isolated from P. aeruginosa PAU5 when exposed to 1-10 mM-CAA. This mutant, P. aeruginosa PAU6, synthesized an inducible A-amidase. CAA-resistance depended upon the simultaneous expression of CAA-inducible amidase and dehalogenase activities.     

Purification and characterization of a dehalogenase from Pseudomonas stutzeri DEH130 isolated from the marine sponge Hymeniacidon perlevis

2-haloacid dehalogenases are enzymes that are capable of degrading 2-haloacid compounds. These enzymes are produced by bacteria, but so far they have only been purified and characterized from terrestrial bacteria. The present study describes the purification and characterization of 2-haloacid dehalogenase from the marine bacterium Pseudomonas stutzeri DEH130. P. Stutzeri DEH130 contained two kinds of 2-haloacid dehalogenase (designated as Dehalogenase I and Dehalogenase II) as detected in the crude cell extract after ammonium sulfate fractionation. Both enzymes appeared to exhibit stereo-specificity with respect to substrate. Dehalogenase I was a 109.9-kDa enzyme that preferentially utilized D-2-chloropropropionate and had optimum activity at pH 7.5. Dehalogenase II, which preferentially utilized L-2-chloropropionate, was further purified by ion-exchange chromatography and gel filtration. Purified Dehalogenase II appeared to be a dimeric enzyme with a subunit of 26.0-kDa. It had maximum activity at pH 10.0 and a temperature of 40 °C. Its activity was not inhibited by DTT and EDTA, but strongly inhibited by Cu²⁺, Zn²⁺, and Co²⁺. The K _m and V _max for L-2-chloropropionate were 0.3 mM and 23.8 μmol/min/mg, respectively. Its substrate specificity was limited to short chain mono-substituted 2-halocarboxylic acids, with no activity detected toward fluoropropionate and monoiodoacetate. This is the first report on the purification and characterization of 2-haloacid dehalogenase from a marine bacterium.     

Regulation of carbon and electron flow in Propionispira arboris: relationship of catabolic enzyme levels to carbon substrates fermented during propionate formation via the methylmalonyl coenzyme A pathway.

A detailed study of the glucose fermentation pathway and the modulation of catabolic oxidoreductase activities by energy sources (i.e., glucose versus lactate or fumarate) in Propionispira arboris was performed. 14C radiotracer data show the CO2 produced from pyruvate oxidation comes exclusively from the C-3 and C-4 positions of glucose. Significant specific activities of glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-bisphosphate aldolase were detected, which substantiates the utilization of the Embden-Meyerhoff-Parnas path for glucose metabolism. The methylmalonyl coenzyme A pathway for pyruvate reduction to propionate was established by detection of significant activities (greater than 16 nmol/min per mg of protein) of methylmalonyl coenzyme A transcarboxylase, malate dehydrogenase, and fumarate reductase in cell-free extracts and by 13C nuclear magnetic resonance spectroscopic demonstration of randomization of label from [2-13C]pyruvate into positions 2 and 3 of propionate. The specific activity of pyruvate-ferredoxin oxidoreductase, malate dehydrogenase, fumarate reductase, and transcarboxylase varied significantly in cells grown on different energy sources. D-Lactate dehydrogenase (non-NADH linked) was present in cells of P. arboris grown on lactate but not in cells grown on glucose or fumarate. These results indicate that growth substrates regulate synthesis of enzymes specific for the methylmalonyl coenzyme A path and initial substrate transformation.     

The microbial degradation of cyclohexanecarboxylic acid by a beta-oxidation pathway with simultaneous induction to the utilization of benzoate

The metabolism of cyclohexanecarboxylic acid by a bacterium, designated PRL W19, follows a pathway involving beta-oxidation of coenzyme A intermediates analogous to the classical oxidation of fatty acids. The organism appears to have the property for the constitutive metabolism of caproic acid, and cell extracts contain high levels of the enzymes required for the functioning of the fatty acid cycle. However, the metabolism of cyclohexanecarboxylic acid requires induction by growth or incubation with an appropriate substrate. Extracts of induced cells contain several enzyme activities which are synthesized in response to the induction process. These enzymes include cyclohexanecarboxyl-CoA synthetase, cyclohexanecarboxyl-CoA dehydrogenase, 1-cyclohexenecarboxyl-CoA hydratase, and trans-2-hydroxycyclohexanecarboxyl-CoA dehydrogenase. A characteristics feature of this organism is that it becomes induced for the metabolism of benzoate and catechol during growth on cyclohexanecarboxylic acid, but benzoate does not appear to be an obligatory intermediate in the metabolism of cyclohexanecarboxylic acid.     

LM cell growth and membrane lipid adaptation to sterol structure

Using a sterol auxotroph of the LM cell mouse fibroblast, we demonstrate that relatively few cholesterol analogues can substitute for cholesterol as a growth factor. The auxotroph grows normally on desmosterol and trans-22-dehydrocholesterol and at reduced rates on dihydrocholesterol, campesterol, and 22,23-dihydrobrassicasterol. It does not grow with beta-sitosterol, stigmasterol, ergosterol, or cis-22-dehydrocholesterol when the sterol is present as sole supplement but does grow at normal rates when the analogue is supplied with suboptimal amounts of cholesterol. Two contrasting types of membrane lipid changes are observed in cells grown on cholesterol analogues. In cells grown with dihydrocholesterol, a marked increase in desaturation and elongation of fatty acids is noted. Conversely, when cells are grown with cis-22-dehydrocholesterol, desaturation and elongation of fatty acids are severely curtailed. Cells grown on alkyl sterols respond like cells grown on cis-22-dehydrocholesterol but in a less pronounced fashion. The effects of sterol substitution in mammalian cells versus in lower eukaryotes are compared, and an explanation for the secondary changes in fatty acid composition in terms of phospholipid phase behavior is suggested.     

Cometabolism of 3,4-dichlorobenzoate by Acinetobacter sp. strain 4-CB1

When Acinetobacter sp. strain 4-CB1 was grown on 4-chlorobenzoate (4-CB), it cometabolized 3,4-dichlorobenzoate (3,4-DCB) to 3-chloro-4-hydroxybenzoate (3-C-4-OHB), which could be used as a growth substrate. No cometabolism of 3,4-DCB was observed when Acinetobacter sp. strain 4-CB1 was grown on benzoate. 4-Carboxyl-1,2-benzoquinone was formed as an intermediate from 3,4-DCB and 3-C-4-OHB in aerobic and anaerobic resting-cell incubations and was the major transient intermediate found when cells were grown on 3-C-4-OHB. The first dechlorination step of 3,4-DCB was catalyzed by the 4-CB dehalogenase, while a soluble dehalogenase was responsible for dechlorination of 3-C-4-OHB. Both enzymes were inducible by the respective chlorinated substrates, as indicated by oxygen uptake experiments. The dehalogenase activity on 3-C-4-OHB, observed in crude cell extracts, was 109 and 44 nmol of 3-C-4-OHB min-1 mg of protein-1 under anaerobic and aerobic conditions, respectively. 3-Chloro-4-hydroxybenzoate served as a pseudosubstrate for the 4-hydroxybenzoate monooxygenase by effecting oxygen and NADH consumption without being hydroxylated. Contrary to 4-CB metabolism, the results suggest that 3-C-4-OHB was not metabolized via the protocatechuate pathway. Despite the ability of resting cells grown on 4-CB or 3-C-4-OHB to carry out all of the necessary steps for dehalogenation and catabolism of 3,4-DCB, it appeared that 3,4-DCB was unable to induce the necessary 4-CB dehalogenase for the initial p-dehalogenation step.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

A Mechanistic Analysis of Enzymatic Degradation of Organohalogen Compounds

Enzymes that catalyze the conversion of organohalogen compounds have been attracting a great deal of attention, partly because of their possible applications in environmental technology and the chemical industry. We have studied the mechanisms of enzymatic degradation of various organic halo acids. In the reaction of L-2-haloacid dehalogenase and fluoroacetate dehalogenase, the carboxylate group of the catalytic aspartate residue nucleophilically attacked the α-carbon atom of the substrates to displace the halogen atom. In the reaction catalyzed by DL-2-haloacid dehalogenase, a water molecule directly attacked the substrate to displace the halogen atom. In the course of studies on the metabolism of 2-chloroacrylate, we discovered two new enzymes. 2-Haloacrylate reductase catalyzed the asymmetric reduction of 2-haloacrylate to produce L-2-haloalkanoic acid in an NADPH-dependent manner. 2-Haloacrylate hydratase catalyzed the hydration of 2-haloacrylate to produce pyruvate. The enzyme is unique in that it catalyzes the non-redox reaction in an FADH₂-dependent manner.