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

Abstract

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 H 2 18 O by use of a large excess of the enzyme with d- or l-2-chloropropionate as a substrate, the lactate produced was labeled with 18 O . 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.