Cloning, Expression and Preliminary Characterization of Novel Haloalkane Dehalogenase DhmA from Mycobacterium avium N85
Jesenska, A., Bartos, M., Czernekova, V., Rychlik, I., Pavlik, I., Damborsky, J.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY 68: 3724-3730 (2002)
Haloalkane dehalogenases are microbial enzymes catalyzing the cleavage of carbon-halogen bond by a hydrolytic mechanism. Until recently, these enzymes have only been isolated from bacteria living in contaminated environments. This report describes the cloning of dehalogenase gene gene dhmA from Mycobacterium avium subsp. avium N85 isolated from swine’s mesenteric lymph node. The dhmA gene has G+C content 68.21 % and codes for a polypeptide 301 amino acids long with calculated molecular mass of 34.7 kDa. The molecular mass of DhmA determined by SDS electrophoresis and gel permeation chromatography is 34.0 and 35.4 kDa, respectively. Many residues essential for dehalogenation reaction are conserved in DhmA, i.e. the putative catalytic triad consists of Asp123, His279 and Asp250; the putative oxyanion hole is made of Glu55 and Trp124. Trp124 should be involved in substrate binding and product (halide) stabilization while the second halide stabilising residue cannot be identified from comparison of DhmA sequence with the sequences of three dehalogenases with known tertiary structure. The haloalkane dehalogenase DhmA shows broad substrate specificity and good activity with priority pollutant 1,2-dichloroethane. Also, DhmA has significantly lower stability compared to other currently known haloalkane dehalogenases. This study confirms the presence of hydrolytic dehalogenase in facultative pathogen M. avium. The presence of dehalogenase-like genes in the genomes of other mycobacteria, including obligatory pathogens Mycobacterium tuberculosis and Mycobacterium bovis, as well as in other bacterial species like Mesorhizobium loti, Xylella fastidiosa, Photobacterium profundum and Caulobacter crescentus, let us speculate that haloalkane dehalogenase have some other function besides catalysis of hydrolytic dehalogenation of halogenated substances.