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 (H2O)-O-18 by use of a large excess of the enzyme with D- or L-2-chloropropionate as a substrate, the lactate produced was labeled with O-18. 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. (C) 2003 Elsevier B.V. All rights reserved.