Dichloromethane dehalogenase of Hyphomicrobium sp. strain DM2

Abstract

Dichloromethane dehalogenase, a highly inducible glutathione-dependent enzyme catalyzing the conversion of dichloromethane into formaldehyde and inorganic chloride, was purified fivefold with 60% yield from Hyphomicrobium sp. strain DM2. The electrophoretically homogeneous purified enzyme exhibited a specific activity of 17.3 mkat/kg of protein. Its pH optimum was 8.5. The enzyme was stable at -20° for at least 6 months. A subunit molecular weight of 33,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration of native dichloromethane dehalogenase yielded a molecular weight of 195,000. Subunit cross-linking with dimethyl suberimidate confirmed the hexameric tertiary structure of the enzyme. Dichloromethane dehalogenase was highly specific for dihalomethanes. Its apparent K(m) values were 30 μM for CH2Cl2, 15 μM for CH2BrCl, 13 μM for CH2Br2, 5 μM for CH2I2, and 320 μM for glutathione. Several chlorinated aliphatic compounds inhibited the dichloromethane dehalogenase activity of the pure enzyme. The K(i) values of the competitive inhibitors 1,2-dichloroethane and 1-chloropropane were 3 and 56 μM, respectively.