Cloning, Biochemical Properties, and Distribution of Mycobacterial Haloalkane Dehalogenases

Author:

Jesenská Andrea1,Pavlová Martina1,Strouhal Michal1,Chaloupková Radka1,Těšínská Iva1,Monincová Marta1,Prokop Zbyněk1,Bartoš Milan2,Pavlík Ivo2,Rychlík Ivan2,Möbius Petra3,Nagata Yuji4,Damborský Jiři1

Affiliation:

1. Loschmidt Laboratories, Masaryk University, Kamenice 5/A4, 625 00 Brno, Czech Republic

2. Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic

3. Federal Research Institute of Animal Health, Naumburger Str. 96a, 07743 Jena, Germany

4. Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai, 980-8577, Japan

Abstract

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°C. The melting temperature assessed by circular dichroism spectroscopy of DmbA is 47°C and DmbB is 57°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.

Publisher

American Society for Microbiology

Subject

Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology

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