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

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