Screening the Mycobacterium tuberculosis H37Rv genomic library for complementation of catabolic defect for cAMP-dependent expression of maltose operon produced the adenylyl cyclase gene (Mtb cya, (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 hydrophobic 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 domain (245 aa) with extensive homology to the catalytic core of eukaryotic adenylyl 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 the catalytic site in Mtb adenylyl cyclase requires an interaction between Arg(43) and Arg(44) residues in the distal cytoplasmic tail and the carboxyl-terminal cytoplasmic domain. Mtb adenylyl cyclase activity at the physiological concentration of ATP (1 mm) was 475 nmol of cAMP/min/mg of membrane protein in the presence of Mn(2+) but only 10 nmol of cAMP/min/mg of membrane protein in the presence of Mg(2+). The physiological significance of the activation of Mtb adenylyl cyclase by Mn(2+) is discussed in view of the presence of manganese transporter protein in mycobacteria and macrophages wherein mycobacteria reside.