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

Abstract

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 C 2 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.DL-2-Haloacid dehalogenase from Pseudomonas sp. strain 113 (DL-DEX) catalyzes the hydrolytic dehalogenation of both enantiomers of 2-haloalkanoic acids to produce the corresponding 2-hydroxyalkanoic acids with inversion of the C 2 configuration (14, 15). DL-DEX is a unique enzyme; it acts on the chiral carbon of the substrate enantiomers, which are used as equivalent substrates. Most other enzymes acting on the chiral carbon of substrates act on a sole enantiomer. Racemases and DL-DEX are the exceptions, although racemases differ markedly from DL-DEX in that they catalyze stereochemical but not chemical conversion of substrates. Racemases are believed to possess a single and common binding site for both substrate enantiomers. Thus, it will be interesting to clarify whether DL-DEX has a common site or distinct ones for each enantiomer in order to compare it with racemases. Different kinds of 2-haloacid dehalogenases showing different stereospecificities have been isolated from several bacterial strains (2, 6, 21). L-2-Haloacid dehalogenases specifically act on L-enantiomers of 2-haloacids to produce the corresponding D-2-hydroxy acids (11, 13, 23). D-2-Haloacid dehalogenase from Pseudomonas putida AJ1 dehalogenates D-enantiomers to form L-2-hydroxy acids (22). Although the two families of dehalogenases act on opposite enantiomers of 2-haloacids, they are similar to each other in that they act exclusively on one particular enantiomer. Thus, DL-DEX differs markedly from them in this respect, although all of them deha...

dl-2-Haloacid Dehalogenase fromPseudomonas sp. 113 Is a New Class of Dehalogenase Catalyzing Hydrolytic Dehalogenation Not Involving Enzyme-Substrate Ester Intermediate

“…This applies to the dehalogenations of both enantiomers of 2-haloalkanoic acids because the results obtained for both enantiomers were virtually the same. We previously reported that DL-DEX 113 has a single and common catalytic site for both L-and D-enantiomers based on a site-directed mutagenesis experiment and kinetic analysis (17). This conclusion is supported by our present data showing that the enzymatic dehalogenations of both enantiomers proceed through the same mechanism as shown in Fig.…”

“…2), and found that it is similar to that of D-DEX from Pseudomonas putida AJ1 (17). We also showed that DL-DEX 113 has a single and common catalytic site for both D-and L-enantiomers based on a site-directed mutagenesis experiment and kinetic analysis (17). In the present study, we analyzed the reaction mechanism of DL-DEX 113 by means of 18 O incorporation experiments, and found that the reaction does not involve the formation of an enzyme-substrate ester intermediate.…”

“…Purification of DL-DEX 113-Recombinant Escherichia coli JM109 cells harboring p4b (1) encoding DL-DEX 113 (17) were cultivated at 37°C for 14 -18 h in a Luria-Bertani medium (1% polypeptone, 0.5% yeast extract, and 1% NaCl, pH 7.0) containing 150 g/ml ampicillin and 0.2 mM isopropyl-1-thio-␤-D-galactoside. The cells were collected by centrifugation, suspended in a 50 mM potassium phosphate buffer (pH 7.0), and disrupted by ultrasonic oscillation at 4°C for 20 min with a Seiko Instruments ultrasonic disintegrator model 7500.…”

“…Thus, DL-DEX 113 is unique in that its reaction does not involve the formation of an ester intermediate. Since D-DEX from P. putida AJ1 (27) shows sequence similarity with DL-DEX 113 (17), the reaction of D-DEX probably proceeds through the mechanism shown in Fig. 1B.…”

“…We previously determined the primary structure of DL-DEX 113 (Fig. 2), and found that it is similar to that of D-DEX from Pseudomonas putida AJ1 (17). We also showed that DL-DEX 113 has a single and common catalytic site for both D-and L-enantiomers based on a site-directed mutagenesis experiment and kinetic analysis (17).…”