Dichloromethane Dehalogenase Research Articles

Evidence for Identical Dichloromethane Dehalogenases in Different Methylotrophic Bacteria

SUMMARY: The dichloromethane dehalogenases of four facultative methylotrophs that mineralize dichloromethane were compared. Similar levels of specific activity and a similar subunit M r of the dehalogenases were observed in crude extracts of the four strains. Immunodiffusion analysis and quantitative immunoprecipitation indicated that the dehalogenases closely resemble each other. This was confirmed when the N-terminal amino acid sequences of the four dichloromethane dehalogenases were found to be identical. Some variation was observed with respect to the specificity of the inducers for dichloromethane dehalogenase. In the two Hyphomicrobium strains examined 1,1-dichloroethane and 1,2-dichloroethane were effective gratuitous inducers whereas these compounds exhibited only a marginal inducing effect in the two Pseudomonas strains. The results of this comparative study are compatible with the view that dichloromethane dehalogenase represents a recently evolved enzyme which was horizontally distributed by gene transfer.

Dichloromethane dehalogenase of Hyphomicrobium sp. strain DM2.

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 degrees C 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 Km values were 30 microM for CH2Cl2, 15 microM for CH2BrCl, 13 microM for CH2Br2, 5 microM for CH2I2, and 320 microM for glutathione. Several chlorinated aliphatic compounds inhibited the dichloromethane dehalogenase activity of the pure enzyme. The Ki values of the competitive inhibitors 1,2-dichloroethane and 1-chloropropane were 3 and 56 microM, respectively.