title: Cloning, biochemical properties, and distribution of mycobacterial haloalkane dehalogenases.
abstract: Haloalkane dehalogenases are enzymes that catalyze the cleavage of the carbon-halogen bond by a hydrolytic mechanism. Genomes of Mycobacterium tuberculosis and M. bovis contain at least two open reading frames coding for the polypeptides showing a high sequence similarity with biochemically characterized haloalkane dehalogenases. We describe here the cloning of the haloalkane dehalogenase genes dmbA and dmbB from M. bovis 5033/66 and demonstrate the dehalogenase activity of their translation products. Both of these genes are widely distributed among species of the M. tuberculosis complex, including M. bovis, M. bovis BCG, M. africanum, M. caprae, M. microti, and M. pinnipedii, as shown by the PCR screening of 48 isolates from various hosts. DmbA and DmbB proteins were heterologously expressed in Escherichia coli and purified to homogeneity. The DmbB protein had to be expressed in a fusion with thioredoxin to obtain a soluble protein sample. The temperature optimum of DmbA and DmbB proteins determined with 1,2-dibromoethane is 45 degrees C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47 degrees C and DmbB is 57 degrees C. The pH optimum of DmbA depends on composition of a buffer with maximal activity at 9.0. DmbB had a single pH optimum at pH 6.5. Mycobacteria are currently the only genus known to carry more than one haloalkane dehalogenase gene, although putative haloalkane dehalogenases can be inferred in more then 20 different bacterial species by comparative genomics. The evolution and distribution of haloalkane dehalogenases among mycobacteria is discussed.
authors.affiliation: Loschmidt Laboratories, Masaryk University, Kamenice 5/A4, 625 00 Brno, Czech Republic. andreaj@chemi.muni.cz
authors.forename: Andrea
authors.initials: A
authors.lastname: Jesenská
authors.forename: Martina
authors.initials: M
authors.lastname: Pavlová
authors.forename: Michal
authors.initials: M
authors.lastname: Strouhal
authors.forename: Radka
authors.initials: R
authors.lastname: Chaloupková
authors.forename: Iva
authors.initials: I
authors.lastname: Tesínská
authors.forename: Marta
authors.initials: M
authors.lastname: Monincová
authors.forename: Zbynek
authors.initials: Z
authors.lastname: Prokop
authors.forename: Milan
authors.initials: M
authors.lastname: Bartos
authors.forename: Ivo
authors.initials: I
authors.lastname: Pavlík
authors.forename: Ivan
authors.initials: I
authors.lastname: Rychlík
authors.forename: Petra
authors.initials: P
authors.lastname: Möbius
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nagata
authors.forename: Jiri
authors.initials: J
authors.lastname: Damborsky
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D009163:Mycobacterium bovis / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D006867:Hydrolases; C046137:haloalkane dehalogenase