Prokaryotes also display an unequalled variety of the universal class III adenylyl cyclases. The abundance of cAMP producing enzymes forms a stark contrast to the presence of only a few putative guanylyl cyclases in prokaryotes. This was subsequently confirmed by sequence alignment studies. Though the functional roles of GCs in prokaryotes are yet to be unraveled, recently Marden et al. and An et al. have identified AZ 960 JAK inhibitor cyclic GMP dependent signaling pathways in bacteria. Comparison of nucleotidyl cyclases has shown that prokaryotic GCs share a close similarity to bacterial ACs. These bacterial ACs in turn resemble mammalian ACs, as shown by several workers. Ma1120, an adenylyl cyclase present in M. avium shares high sequence similarity with GCs, so this raised the question as to whether Ma1120 could be converted to GC. Many have tried to use mutational analysis and bioinformatics to understand the evolution of these nucleotidyl cyclases and the conservation of certain amino acid residues at the active sites. However two groups have reported the conversion of GC to AC by replacing two crucial amino acids at the substrate binding site – namely E to K and C to D.This is probably due to the fact that the crystal structures of mammalian adenylyl cyclases helped to understand theinteractions of K and D with the substrate. This has not been the case with the guanylyl cyclases where the conservation and interaction of specific residues with GTP is not as clearly defined as in the case of the adenylyl cyclases. The CYG12 guanylyl cyclase from Chlamydomonas reinhardtii contains an E-C pair typical of mammalian guanylyl cyclases while the bacterial Cya2 guanylyl cyclase has an E-G pair. Changing the substrate binding residues has often led to a diminishing of activity rather than a conversion from adenylyl cyclase to guanylyl cyclase. Multiple sequence alignment of Ma1120 cyclase domain with representative cyclase domains of ACs and GCs shows that the substrate binding residues, lysine and aspartate are conserved in ACs across species. In GCs, glutamate is present instead of K while in place of aspartate, one observes a variety of seemingly unrelated amino acid residues that include cysteine, serine, threonine, histidine, alanine or glycine. In this paper we address the question – how do the amino acid residues at the second substrate binding site dictate the nucleotidyl triphosphate preference of the enzyme. For this purpose, Ma1120 having K and D was used as a model to study the consequences of replacing ATP specifying residues with GTP specifying residues. This would help understand how preference for substrates could have evolved. In this study the systematic replacement of ATP specifying amino acid residues to GTP specifying ones, has provided information on how these residues interact with the substrate. As in many other ACs, the first substrate binding residue K probably interacts through H-bonding with the N1 of ATPwhile the second substrate binding amino acid residue, aspartate hydrogen bonds to 6-amino group of ATP. However, when K is mutated to E, this hydrogen bond between the e-amino group of K and the N1 and the 6-amino group of ATP may no longer be possible. The low Km value could be due to an improper orientation of ATP in the active site, leading to a decrease in turnover number as reflected by the kcat value. On the contrary when GTP is the substrate, E can interact through hydrogen bonding with the 2-amino group of GTP which could be in an orientation conducive for catalysis thus resulting in an increase in catalytic turnover. The adenylyl cyclase:ATP analogue complex crystal structure available in the data base.