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

Abstract

18 O of the solvent directly attacks the ␣-carbon of 2-haloalkanoic acid to displace the halogen atom. This is the first example of an enzymatic hydrolytic dehalogenation that proceeds without producing an ester intermediate.Various enzymes catalyzing hydrolytic dehalogenation of organohalogen compounds have been isolated and characterized (1-3). These enzymes include 2-haloacid dehalogenases (EC 3.8.1.2), haloacetate dehalogenases (EC 3.8.1.3), haloalkane dehalogenases (EC 3.8.1.5), and 4-chlorobenzoyl-CoA dehalogenases (EC 3.8.1.6). 2-Haloacid dehalogenases are further classified into three groups based on their substrate specificities (4). L-2-Haloacid dehalogenase (L-DEX) 1 specifically acts on L-2-haloalkanoic acids, and the corresponding D-2-hydroxyalkanoic acids are produced. D-2-Haloacid dehalogenase (D-DEX) catalyzes the conversion of D-2-hydroxyalkanoic acids into L-2-hydroxyalkanoic acids. DL-2-Haloacid dehalogenase (DL-DEX) dehalogenates both D-and L-2-haloalkanoic acids, and the corresponding L-and D-2-hydroxyalkanoic acids are produced. DL-DEX is similar to racemases and epimerases in that it acts indiscriminately on the chiral center of both D-and L-enantiomers. However, this enzyme is unique in that it catalyzes a chemical conversion on the chiral centers of both enantiomers. Thus far, the reaction mechanisms of L-DEX from Pseudomonas sp. YL (L-DEX YL) (5-7), haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (8 -10), and 4-chlorobenzoylCoA dehalogenases from Pseudomonas sp. strain CBS3 (11,12) and Arthrobacter sp. 4-CB1 (13) have been analyzed. Their reactions proceed as shown in Fig. 1A. Each of these dehalogenases has an acidic amino acid residue whose carboxylate group attacks the carbon atom of the substrate to which the halogen atom is bound. Asp 10 of L-DEX YL, Asp 124 of haloalkane dehalogenase from X. autotrophicus GJ10, and Asp 145 of 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp. strain CBS3 were identified to play this essential role for respective enzymes. The ester intermediates produced in the course of these reactions are subsequently hydrolyzed releasing the products and restoring the carboxylate groups of the enzymes. These were confirmed by chemical modification, sitedirected mutagenesis, mass spectrometry, and x-ray crystallographical analysis (5-13).DL-DEXs have been purified from Pseudomonas sp. 113 (DL-DEX 113) (14), Pseudomonas putida PP3 (15), and Rhizobium sp. (16). However, none of the reaction mechanisms of these DL-DEXs have been studied, and it is unknown whether the reaction mechanism of DL-DEX is similar to that of other halidohydrolases (dehalogenases that catalyze the hydrolytic dehalogenation). 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). In the present study, we analyze...

Microbial dehalogenation

“…113, incorporation of 18 O into the enzyme was not detected. Thus, Nardi-Dei et al [9] proposed that the dehalogenase directly activates a water molecule to attack the α carbon of 2-haloalkanoic acid, thereby displacing the halogen atom. It has not yet been established which amino acids are involved in the dehalogenation, although a number of candidates have been highlighted by mutagenesis experiments.…”

D-Specific Dehalogenases, a Review

“…3). nardi-Dei et al (25) observed that an enzyme activated water molecule directly attacks the α-carbon of the 2-haloalkanoic acid to displace the halogen atom. this was the first example of an enzymatic hydrolytic dehalogenation that proceeds without producing an ester intermediate.…”

“…1) (9) showed that DehD and hadD share 27% identity. Sequences ile2 [8], leu4 [10], Pro9 [15], ile15 [21], Arg16 [22], Val18 [24], Pro19 [25], Glu20 [26], his21 [27], Ala23 [29], Glu26 [32], leu27 [33] the amino acid compositions of DehD (265 residues) and hadD (301 residues) are compared in Table 3. there are marked differences in molecular weight, theoretical pi value, aliphatic index, and the grand average hydropathy value.…”

A Mechanistic Analysis of Enzymatic Degradation of Organohalogen Compounds

“…The mechanism was further studied by 18 O-incorporation experiments. 26) When a single turnover reaction of DL-DEX 113 was carried out using a large excess of the enzyme in H 2 18 O with a 10-times smaller amount of the substrate, D-or L-2-chloropropionate, the major product was found to be 18 O-labeled lactate. Mass spectrometric analysis of the enzyme after a multiple turnover reaction in H 2 18 O revealed that 18 O was not incorporated into the enzyme during the reaction.…”

“…Considering the sequence similarity between DL-2-haloacid dehalogenase and D-2-haloacid dehalogenase, the reaction catalyzed by D-2-haloacid dehalogenase probably proceeds through the same mechanism as that of DL-2-haloacid dehalogenase. 25,26) Analysis of solvent deuterium and chlorine kinetic isotope effects on the reactions catalyzed by DL-DEX 113 for D-and L-2-chloropropionate were also carried out. 30) It was found that a step preceding dehalogenation is partly rate-limiting in the case of D-2-chloropropionate, and that the overall reaction rates are controlled by different steps in the catalysis of D-and L-2-chloropropionate.…”