Publication details

Validation of Putative Enzymes from Genomic Projects: Confirmation of Dehalogenation ARv2ctivity for Mycobacterial Protein 579

Title in English Validation of Putative Enzymes from Genomic Projects: Confirmation of Dehalogenation Activity for Mycobacterial Protein Rv2579
Authors

MONINCOVÁ Marta NAGATA Yuji PROKOP Zbyněk MARVANOVÁ Soňa SÝKOROVÁ Jana TSUDA Masataka DAMBORSKÝ Jiří

Year of publication 2003
Type Article in Proceedings
Conference Management and Control of (Undesirable) Microorganisms, IBBS/IBRG Meeting Abstracts
MU Faculty or unit

Faculty of Science

Citation
Field Biochemistry
Keywords dehalogenase; halogenated hydrocarbons; site-directed mutagenesis
Description Haloalkane dehalogenases are the microbial enzymes which catalyse cleavage of the carbon-halogen bond. There are many microorganisms capable to dehalogenate halogenated hydrocarbons. Hydrolytic dehalogenating activity was detected also with a number of different mycobacteria. The putative haloalkane dehalogenase gene rv2579 was found in the complete genomic sequence of the human pathogen Mycobacterium tuberculosis H37Rv. Initially, computer modelling and site-directed mutagenesis experiments were conducted to confirm dehalogenating activity for Rv2579. The homology model of protein Rv2579 was compared with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26. This analysis revealed that 6 of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. To characterize the effect of replacement of these six residues, mutations were introduced cumulatively into six amino acid residues of LinB. The sixfold mutant, which was supposed to have the active site of Rv2579, exhibited dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase family. Consequently the rv2579 gene was cloned from Mycobacterium bovis into Escherichia coli. Rv2579 protein was overproduced and purified to homogeneity. Rv2579 shows hydrolytic dehalogenating activity, further confirming the conclusions of the site-directed mutagenesis study. Detailed comparison of the catalytic properties of sixfold LinB mutant and Rv2579 is in progress. This comparison will validate applicability of reconstruction of an active site of an enzyme with putative function in an enzyme with known function.

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