Characterization of phylogenetically distant members of the adenylyl cyclase family from mycobacteria: Rv1647 from M. tuberculosis and its ortholog ML1399 from M. leprae

Abstract

Analysis of the genome sequence of Mycobacterium tuberculosis H37Rv has identified 16 genes that are similar to the mammalian adenylyl and guanylyl cyclases. Rv1647 was predicted to be an active adenylyl cyclase but its position in a phylogenetically distant branch from the other enzymes characterized so far from M. tuberculosis, makes it an interestingly divergent nucleotide cyclase to study. In agreement with its divergence at the sequence level from other nucleotide cyclases, cloning, expression and purification of Rv1647 revealed differences in its biochemical properties from the earlier characterized Rv1625c adenylyl cyclase. Adenylyl cyclase activity of Rv1647 was activated by detergents but was resistant to high concentrations of salt. Mutations of substrate specifying residues to those present in guanylyl cyclases failed to convert the enzyme to a guanylyl cyclase, but did not alter its oligomeric status. Orthologs of Rv1647 could be found in M. leprae, M. avium and M. smegmatis. The ortholog from M. leprae (ML1399) was cloned, protein expressed, purified and shown biochemically to be an adenylyl cyclase, thus representing the first adenylyl cyclase to be described from M. leprae. Importantly, Western blot analysis of subcellular fractions from M. tuberculosis and M. leprae revealed that Rv1647 and ML1399 gene products were expressed in these bacteria respectively. Additionally, M. tuberculosis was also found to express the Rv1625c adenylyl cyclase, suggesting that multiple adenylyl cyclase proteins may be expressed simultaneously in this organism. These results suggest that Class III cyclaselike gene products are likely to have an important role to play in the physiology and perhaps the pathology of these medically important bacteria.