Eukaryotic-like adenylyl cyclases in Mycobacterium tuberculosis H37Rv - Cloning and characterization

Screening the Mycobacterium tuberculosis H37Rv genomic library for compleme ntation of catabolic defect for cAMP-dependent expression of maltose operon produced the adenylyl cyclase gene (Mtb cya, GenBank(TM) accession no. AF0 17731 (1997)) annotated later as Rv1625c (Cole, S. T., Brosch, R., Parkhill , J., Garnier, T., Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S., Barry, C. E., III, et al. (1998) Nature 393, 537-544). The deduced amino acid (aa) sequence (443 aa) encoded by Mtb cya contains a single hyd rophobic domain of six transmembrane helices (152 aa) in the amino-terminal half of the protein. Flanking this domain are an arginine-rich (17%) amino -terminal cytoplasmic tail (46 aa) and a carboxyl-terminal cytoplasmic doma in (245 aa) with extensive homology to the catalytic core of eukaryotic ade nylyl cyclases. Site-directed mutagenesis of Arg(43) and Arg(44) to alanine /glycine showed a loss of adenylyl cyclase activity, whereas mutagenesis to lysine restored the activity. Hence it is proposed that the formation of t he catalytic site in Mtb adenylyl cyclase requires an interaction between A rg(43) and Arg(44) residues in the distal cytoplasmic tail and the carboxyl -terminal cytoplasmic domain. Mtb adenylyl cyclase activity at the physiolo gical concentration of ATP (1 mm) was 475 nmol of cAMP/min/mg of membrane p rotein in the presence of Mn2+ but only 10 nmol of cAMP/min/mg of membrane protein in the presence of Mg2+. The physiological significance of the acti vation of Mtb adenylyl cyclase by Mn2+ is discussed in view of the presence of manganese transporter protein in mycobacteria and macrophages wherein m ycobacteria reside.