Reconstruction of Mycobacterial Dehalogenase Rv2579 by Cumulative Mutagenesis of Haloalkane Dehalogenase LinB

Abstract

The homology model of protein Rv2579 from Mycobacterium tuberculosis H37Rv was compared with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26, and this analysis revealed that 6 of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. To characterize the effect of replacement of these six amino acid residues, mutations were introduced cumulatively into the six amino acid residues of LinB. The sixfold mutant, which was supposed to have the active site of Rv2579, exhibited haloalkane dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase protein family.

Cloning, Biochemical Properties, and Distribution of Mycobacterial Haloalkane Dehalogenases

“…Currently, Mycobacterium is the only known genus expressing two such different enzymes within a single species. Comparison of the kinetic constants of wild-type DmbA with the previously constructed sixfold mutant LinB validated the earlier proposal of the catalytic function of this protein established from computer modeling and site-directed mutagenesis (32). Cloned genes could be used for DNA shuffling studies to reconstruct novel catalysts for degradation of important environmental pollutants, e.g., 1,2-dichloroethane and 1,2,3-trichloropropane, which was not possible previously due to the low homology of available dehalogenase genes.…”

“…The specific activity of DmbA under optimal conditions is 0. (32). In the present study, the pure DmbA was obtained and its dehalogenase activity was confirmed.…”

“…To confirm the prediction and test a general concept of the functional characterization of proteins from genome projects using site-directed mutagenesis, we introduced mutations to the active site of the homologous protein LinB (32). The constructed sixfold mutant of LinB mimicking the active site of the protein encoded by rv2579, dbmA, showed clear dehalogenating activity.…”

“…In a previous study, we compared the homology model of protein Rv2579 with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 (25) and found that 6 of 19 amino acid residues that form an active site and entrance tunnel are different between LinB and Rv2579 (32). Mutations were introduced cumulatively into the six amino acid residues of LinB resulting in a protein mutant which was designed to have an active site of Rv2579.…”

Two Rhizobial Strains,Mesorhizobium lotiMAFF303099 andBradyrhizobium japonicumUSDA110, Encode Haloalkane Dehalogenases with Novel Structures and Substrate Specificities

“…Comparison of the kinetic mechanisms of DhlA, DhaA, and LinB showed that the overall reaction mechanisms are similar but that the rate-limiting steps differ, i.e., halide release in the case of DhlA (44), liberation of an alcohol in the case of DhaA (4), and hydrolysis of an alkyl-enzyme intermediate in the case of LinB (41). Partial improvement in the catalytic properties and modification of the substrate specificities of haloalkane dehalogenases by rational design (5,34) and directed evolution approaches (3,40) have recently been reported. However, it remains difficult to construct mutant enzymes with entirely new capabilities using only protein-engineering techniques, and therefore, the isolation of new family members is still desirable.…”