Class III adenylyl cyclases usually possess six highly conserved catalytic residues. Deviations in these canonical amino acids are observed in several putative adenylyl cyclase genes as apparent in several bacterial genomes. This suggests that a variety of catalytic mechanisms may actually exist. The gene Rv0386 from Mycobacterium tuberculosis codes for an adenylyl cyclase catalytic domain fused to an AAA‐ATPase and a helix‐turn‐helix DNA‐binding domain. In Rv0386, the standard substrate, adenine‐defining lysine‐aspartate couple is replaced by glutamine‐asparagine. The recombinant adenylyl cyclase domain was active with a Vmax of 8 nmol cAMP·mg−1·min−1. Unusual for adenylyl cyclases, Rv0386 displayed 20% guanylyl cyclase side‐activity with GTP as a substrate. Mutation of the glutamine‐asparagine pair either to alanine residues or to the canonical lysine‐aspartate consensus abolished activity. This argues for a novel mechanism of substrate selection which depends on two noncanonical residues. Data from individual and coordinated point mutations suggest a model for purine definition based on an amide switch related to that previously identified in cyclic nucleotide phosphodiesterases.

The class IIIa adenylyl cyclase (AC) Rv1625c from Mycobacterium tuberculosis forms homodimers with two catalytic centres, whereas the Paramecium guanylyl and mammalian ACs operate as pseudoheterodimers with one catalytic centre. The functional and structural relationship of the catalytic domains of these related class III cyclases was investigated. Point mutations introduced into Rv1625c to engineer a forskolinbinding pocket created a single heterodimeric catalytic centre, yet did not result in forskolin activation. Chimerization of these Rv1625c point mutants with corresponding mammalian AC domains was impossible. However, it was successful using a complemental Paramecium guanylyl cyclase domain and resulted in an AC. The data signify a divergence of structural and functional evolution in class III Acs.