Cyclic AMP, nucleotide cyclases and phosphodiesterases insights from computational, biochemical and functional studies in mycobacteria

Abstract

Cyclic nucleotides (3 ,5 -cyclic AMP and cGMP) were among the first second messengers to be identified, and their roles in diverse signalling pathways have since been widely studied. Nucleotide cyclases (which synthesize cyclic nucleotides) and phosphodiesterases (which degrade them) have been biochemically and structurally characterized. Both enzyme families are divided into several classes based on primary amino acid sequence. Six classes of nucleotide cyclases and three classes of cNMP phosphodiesterases are currently recognized.

Class III nucleotide cyclases have the widest phyletic distribution, constituting the “Universal Class,” and include all known guanylyl cyclases. This thesis focuses mainly on Class III cyclases.

Distribution and Domain Architecture Eukaryotic Class III cyclases include:

Mammalian membrane-bound adenylyl cyclases (12 transmembrane regions, two cyclase domains).

Soluble adenylyl cyclases (two cyclase domains with an ATPase domain).

Receptor guanylyl cyclases (single transmembrane, kinase-homology, and cyclase domains).

Soluble guanylyl cyclases (haeme-binding and cyclase domains).

Protist guanylyl cyclases (12 or 22 transmembrane helices, two cyclase domains).

Bacterial Class III cyclases are fused to diverse domains such as histidine kinase/receiver domains, GAF domains, FHA domains, PAS domains, and tetratricopeptide repeats.

Computational mining of bacterial and archaeal genomes (129 genomes analysed) identified 193 proteins in 29 species. Nitrogen-fixing -proteobacteria (Mesorhizobium, Bradyrhizobium, Sinorhizobium) and actinobacteria (Mycobacterium) contained multiple cyclases.

Functional Classification Based on mammalian adenylyl cyclase crystal structures, bacterial cyclase domains were classified as C1-like or C2-like.

Proteins with two Class III domains were identified in bacteria, representing ancestral candidates.

Substrate specificity was predicted:

Adenylyl cyclases: lysine-aspartate pair.

Guanylyl cyclases: glutamate-cysteine pair.

Mycobacterial Cyclases Mycobacterium tuberculosis contains 17 cyclases; M. leprae (with extensive gene loss) retains 4 functional cyclases.

Comparative genomics identified orthologues across actinobacteria.

RT-PCR confirmed expression of most cyclases in M. tuberculosis.

Biochemical characterization focused on Rv1625c, Rv1120c/Mai120, and Rv1647/orthologues.

Rv1625c:

Six transmembrane helices followed by a cyclase domain.

Substrate specifying residues (lysine-aspartate) mutated to glutamate-cysteine abolished activity and altered oligomeric state.

Detected in plasma membrane fraction of M. tuberculosis.

Rv1120c/Mai120:

Rv1120c predicted pseudogene in M. tuberculosis.

Functional orthologue Mai120 in M. avium characterized as robust adenylyl cyclase.

Rv1647/ML1399/Sml647:

Highly active adenylyl cyclase with orthologues in M. leprae, M. avium, M. smegmatis.

Activity dependent on pH, detergents, or salt.

Mutational studies confirmed catalytic mechanism similar to mammalian cyclases.

Deletion of Sml647 in M. smegmatis altered cAMP response under acid stress.

Phosphodiesterases (PDEs) Class III PDEs belong to the metallo-phosphoesterase superfamily.

Using HMM profiles, 129 putative PDEs were identified, including Rv0805 in M. tuberculosis.

Rv0805 characterized as a Mn² -dependent dimeric PDE hydrolysing both cAMP and cGMP.

Overexpression reduced intracellular cAMP levels in E. coli and M. smegmatis.

Conclusion This thesis combines bioinformatic mining and biochemical characterization to advance understanding of cAMP synthesis and degradation in mycobacteria. It establishes the foundation for studying cyclic nucleotide signalling in pathogenic mycobacteria and other bacteria.