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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2002, p. 3724–3730 0099-2240/02/$04.00 0 DOI: 10.1128/AEM.68.8.3724–3730.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Vol. 68, No. 8

Cloning and Expression of the Haloalkane Dehalogenase Gene dhmA from Mycobacterium avium N85 and Preliminary Characterization of DhmA
Andrea Jesenska,1 Milan Bartos,2 VladimıCzernekova,1,3 Ivan Rychlı 2 Ivo Pavlı 2 ´ ˇ ´ra ´ ´k, ´k, and Jir´ Damborsky1* ˇı ´
National Centre for Biomolecular Research1 and Department of Biochemistry,3 Faculty of Science, Masaryk University, Kotlarska 2, 611 37 Brno, and Veterinary Research Institute, Hudcova 70, 621 32 Brno,2 Czech Republic
Received 22 January 2002/Accepted 18 April 2002

Haloalkane dehalogenases are microbial enzymesthat 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 polypeptidethat 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, andAsp250, 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 goodactivity 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 Mycobacteriumbovis, 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 aliphaticcompounds, 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 havealready 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, 27–30, 40). Only recently have the hydrolytic dehalogenation activities of several species of the genus Mycobacterium isolated from clinical materialbeen 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 H37Rv resulting from a BLAST search of genetic databases. Now many new dehalogenase-like genes can be identified in the genomes of various bacteria by database searches (Table 1). If...
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