Cloning and Expression of the Haloalkane Dehalogenase GenedhmAfromMycobacterium aviumN85 and Preliminary Characterization of DhmA

Abstract

Haloalkane dehalogenases are microbial enzymes that catalyze cleavage of the carbon-halogen bond by a hydrolytic mechanism. Until recently, these enzymes have been isolated only from bacteria living in contaminated environments. In this report we describe cloning of the dehalogenase gene dhmA from Mycobacterium avium subsp. avium N85 isolated from swine mesenteric lymph nodes. The dhmA gene has a G؉C content of 68.21% and codes for a polypeptide that is 301 amino acids long and has a calculated molecular mass of 34.7 kDa. The molecular masses of DhmA determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography are 34.0 and 35.4 kDa, respectively. Many residues essential for the dehalogenation reaction are conserved in DhmA; the putative catalytic triad consists of Asp123, His279, and Asp250, and the putative oxyanion hole consists of Glu55 and Trp124. Trp124 should be involved in substrate binding and product (halide) stabilization, while the second halide-stabilizing residue cannot be identified from a comparison of the DhmA sequence with the sequences of three dehalogenases with known tertiary structures. The haloalkane dehalogenase DhmA shows broad substrate specificity and good activity with the priority pollutant 1,2-dichloroethane. DhmA is significantly less stable than other currently known haloalkane dehalogenases. This study confirms that a hydrolytic dehalogenase is present in the facultative pathogen M. avium. The presence of dehalogenase-like genes in the genomes of other mycobacteria, including the obligate pathogens Mycobacterium tuberculosis and Mycobacterium bovis, as well as in other bacterial species, including Mesorhizobium loti, Xylella fastidiosa, Photobacterium profundum, and Caulobacter crescentus, led us to speculate that haloalkane dehalogenases have some other function besides catalysis of hydrolytic dehalogenation of halogenated substances.Haloalkane dehalogenases catalyze hydrolytic cleavage of carbon-halogen bonds in halogenated aliphatic compounds, leading to the formation of primary alcohols, halide ions, and protons. These enzymes are potentially useful for cleaning up contaminated subsurfaces (32) and for processing by-products of chemical syntheses (33). Haloalkane dehalogenases can serve as a model system for studies of the evolution and distribution of degradation enzymes in the environment since many of these enzymes have already been isolated from different bacterial species originating from geographically distinct areas (26). Haloalkane dehalogenases have primarily been isolated from bacteria colonizing environments contaminated by halogenated substances (12,16,22,(27)(28)(29)(30)40). Only recently have the hydrolytic dehalogenation activities of several species of the genus Mycobacterium isolated from clinical material been reported (14). Motivation for the search for haloalkane dehalogenases in clinical samples of mycobacteria came from the identification of dehalogenase-like genes in the genome of Mycobacterium tuberculosis...

Biochemical Characterization of a Novel Haloalkane Dehalogenase from a Cold-Adapted Bacterium

“…This property is unique and has not been observed for any other HLD so far. The highest activities of other related enzymes lay in the temperature interval between 40 and 50°C (15,(18)(19)(20)(21)26). Another important finding is that the DpcA enzyme retained almost 27% of its maximal activity at 5°C.…”

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

“…It was recently demonstrated that Mycobacterium tuberculosis H37Rv, the complete genome of which has been sequenced (7), possesses chromosomal genes encoding putative haloalkane dehalogenases (11). Hydrolytic dehalogenation was detected in a number of different mycobacteria (18), and haloalkane dehalogenase DhmA from M. avium N85 has been partially characterized (19). Furthermore, we could find dehalogenase-like open reading frames (ORFs) on the genomes of more than 20 bacterial species.…”

“…The dehalogenase activity of the purified His-tagged DbjA protein in the presence of 18 halogenated compounds was measured spectrophotometrically ( Table 2). The relative activity data measured for DbjA with 18 different halogenated substrates were complemented with the data collected for other enzymes from the literature, i.e., DhlA (12), DhaA (12), LinB (12), and DhmA (19), and also with the data obtained using a crude extract of E. coli expressing the Trx-DmlA fusion protein FIG. 3.…”

Biochemical characterization of two haloalkane dehalogenases: DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea

“…[17][18][19] This substrate specificity profile is characteristic of substrate specificity group (SSG)-IV; by contrast, DhlA is categorized in SSG-I. Both DmbB from Mycobacterium bovis 18 and DhmA from Mycobacterium avium N85 21 were categorized in the HLD-I subfamily but their substrate specificities were not reported due to expression and stability problems. The substrate specificity and activity of HLDs have been rationalized by comparing structural differences in the tunnel(s) that provide access to the active site from the surface of the enzyme.…”