Mycobacteria as a source of haloalkane dehalogenases: Identification of the catalytic pentad within the structure of DhmA from Mycobacterium avium N85

 

Andrea Jesenská1,2, Martina Pavlová1, Martin Klvaňa1, Radka Chaloupková1, Marta Monincová1, Takashi Sato3, Masataka Tsuda3, Yuji Nagata3, Zbyněk Prokop1,2 and Jiří Damborský1

 

1 Loschmidt Laboratories, Faculty of Science, Masaryk University, Kamenice 5/A4, Brno, 611 37, Czech Republic

2 National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5/A4, Brno, 611 37, Czech Republic

3 Department of Environmental Life Science, Graduate School of Life Sciences, Tohoku University, Katahira, Sendai 980-8577, Japan

 

Haloalkane dehalogenases belonging to a/b-hydrolase superfamily are microbial enzymes that catalyze hydrolytic cleavage of carbon-halogen bond. This reaction plays a key role in biodegradation of halogenated aliphatic compounds. Haloalkane dehalogenases act on broad range of halogenated aliphatic compounds. These enzymes were isolated mainly from bacteria colonizing contaminated environment.

Comparison of known haloalkane dehalogenases with sequences provided by genomic projects showed that mycobacteria belong to valuable source of new putative haloalkane dehalogenases. Five haloalkane dehalogenases from saprophytic and tissue-colonizing mycobacteria were cloned: dhmA from Mycobacterium avium N85 [1], dmsA from Mycobacterium smegmatis CCM4622, and dmbA, dmbB [2] and dmbC, all from Mycobacterium bovis 5033/66. The proteins encoded by the genes were expressed using different expression systems and purified to homogeneity by metal affinity chromatography. The biochemical characterization of mycobacterial haloalkane dehalogenases showed that they differ in pH, temperature optimum, melting temperature and substrate specificity.

Haloalkane dehalogenase DhmA is the best characterized from the structural point view. Catalytic pentad consists of catalytic triad and two halide-stabilizing amino acid residues. The catalytic triad D123, H279 and D250 was deduced from sequence comparison. Amino acid W124 was identified as the first halide-stabilizing residue. The second halide-stabilizing residue W164 was predicted based on homology modeling. Five single-point mutants D123A, W124L, W164L, D250A and H279A were constructed, expressed in Rhodococcus erythropolis IAM1399 and purified to homogeneity. None of the DhmA mutant showed dehalogenating activity confirming their essential role in catalysis. A reaction mechanism was proposed for DhmA and compared with the mechanism of other family members.

 

[1] A. Jesenska, M. Bartos, V. Czernekova, I. Rychlik, I. Pavlik & J. Damborsky: Appl. Environ. Microbiol., 68 (2002) 3724-3730.

 

[2] A. Jesenska, M. Pavlova, M. Strouhal, R. Chaloupkova, I. Tesinska, M. Monicova, Z. Prokop, M. Bartos, I. Pavlik, I. Rychlik, P. Mobius, Y. Nagata, & J. Damborsky, Appl. Environ. Microbiol., 71 (2005) 6736-674.