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J Biol Chem, Vol. 274, Issue 30, 20977-20981, July 23, 1999

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

Vincenzo Nardi-Dei, Tatsuo Kurihara, Chung Park, Masaru Miyagi^¶, Susumu Tsunasawa^¶, Kenji Soda, and Nobuyoshi Esaki

From the  Laboratory of Microbial Biochemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan and the ^¶ Biotechnology Research Laboratories, Takara Shuzo Co., Ltd., Kusatsu, Shiga 525-0055, Japan

DL-2-Haloacid dehalogenase from Pseudomonas sp. 113 (DL-DEX 113) catalyzes the hydrolytic dehalogenation of D- and L-2-haloalkanoic acids, producing the corresponding L- and D-2-hydroxyalkanoic acids, respectively. Every halidohydrolase studied so far (L-2-haloacid dehalogenase, haloalkane dehalogenase, and 4-chlorobenzoyl-CoA dehalogenase) has an active site carboxylate group that attacks the substrate carbon atom bound to the halogen atom, leading to the formation of an ester intermediate. This is subsequently hydrolyzed, resulting in the incorporation of an oxygen atom of the solvent water molecule into the carboxylate group of the enzyme. In the present study, we analyzed the reaction mechanism of DL-DEX 113. When a single turnover reaction of DL-DEX 113 was carried out with a large excess of the enzyme in H₂¹⁸O with a 10 times smaller amount of the substrate, either D- or L-2-chloropropionate, the major product was found to be ¹⁸O-labeled lactate by ionspray mass spectrometry. After a multiple turnover reaction in H₂¹⁸O, the enzyme was digested with trypsin or lysyl endopeptidase, and the molecular masses of the peptide fragments were measured with an ionspray mass spectrometer. No peptide fragments contained ¹⁸O. These results indicate that the H₂¹⁸O 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.

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