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Applied and Environmental Microbiology, November 2005, p. 6736-6745, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.6736-6745.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Cloning, Biochemical Properties, and Distribution of Mycobacterial Haloalkane Dehalogenases

Andrea Jesenská,1* Martina Pavlová,1 Michal Strouhal,1 Radka Chaloupková,1 Iva Tesínská,1 Marta Monincová,1 Zbynek Prokop,1 Milan Bartos,2 Ivo Pavlík,2 Ivan Rychlík,2 Petra Möbius,3 Yuji Nagata,4 and Jiri Damborsky1

Loschmidt Laboratories, Masaryk University, Kamenice 5/A4, 625 00 Brno, Czech Republic,1 Veterinary Research Institute, Hudcova 70, 621 32 Brno, Czech Republic,2 Federal Research Institute of Animal Health, Naumburger Str. 96a, 07743 Jena, Germany,3 Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai, 980-8577, Japan4

Received 6 May 2005/ Accepted 1 July 2005

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.

* Corresponding author. Mailing address: Loschmidt Laboratories, Masaryk University, Kamenice 5/A4, 625 00 Brno, Czech Republic. Phone: 420-5-49494694. Fax: 420-5-49492556. E-mail: andreaj{at}chemi.muni.cz.

Applied and Environmental Microbiology, November 2005, p. 6736-6745, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.6736-6745.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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