Molecular cloning, expression, purification and functional characterization of Rv1588c of Mycobacterium tuberculosis

Biotin is an essential cofactor for biotin dependent enzymes necessary for synthesis of fatty acids including mycolic acids, an essential component of cell wall of Mycobacterium tuberculosis. Biotin is synthesized by the genes located in bio operons which are fairly conserved in mycobacteria. Biotin synthesis is controlled intracellularly in Mycobacterium smegmatis by the gene bioQ, located opposite to bioB. Such locations of biotin regulators are found to be conserved among various mycobacteria species. In M. tuberculosis, rv1588c is such an uncharacterized gene, located opposite to bioB which shares limited similarity with DNA-binding region of BioQ of M. smegmatis, a TetR-type protein. A highly similar M. smegmatis-like BioQ binding site is also present within the bioB promoter of M. tuberculosis. This suggests that Rv1588c might be the BioQ for M. tuberculosis. The present study reports in-silico and functional characterization of Rv1588c. The structure of Rv1588c has been partially modelled and validated computationally. The N-terminal domain of Rv1588c is found to be structurally similar with DNA-binding domains of several transcriptional regulators. rv1588c gene has been cloned, expressed, and purified. Limited proteolysis study revealed presence of C-terminal domain in Rv1588c which indicates presence of a two-domain structure, like those of canonical transcriptional regulators. Rv1588c forms significant amount of dimers in solution, mediated by cysteine residues. Purified Rv1588c binds with bioB promoter region in-vitro. This suggests that Rv1588c is probably the transcriptional regulator that might control the expression of bioB of M. tuberculosis.