Evolutionary Adaptation of the Essential tRNA Methyltransferase TrmD to the Signaling Molecule 3′,5′-cAMP in Bacteria

Abstract

The nucleotide signaling molecule 3′,5′-cyclic adenosine monophosphate (3′,5′-cAMP) plays important physiological roles, ranging from carbon catabolite repression in bacteria to mediating the action of hormones in higher eukaryotes, including human. However, it remains unclear whether 3′,5′-cAMP is universally present in the Firmicutes group of bacteria. We hypothesized that searching for proteins that bind 3′,5′-cAMP might provide new insight into this question. Accordingly, we performed a genome-wide screen and identified the essential Staphylococcus aureus tRNA m1G37 methyltransferase enzyme TrmD, which is conserved in all three domains of life as a tight 3′,5′-cAMP-binding protein. TrmD enzymes are known to use S-adenosyl-l-methionine (AdoMet) as substrate; we have shown that 3′,5′-cAMP binds competitively with AdoMet to the S. aureus TrmD protein, indicating an overlapping binding site. However, the physiological relevance of this discovery remained unclear, as we were unable to identify a functional adenylate cyclase in S. aureus and only detected 2′,3′-cAMP but not 3′,5′-cAMP in cellular extracts. Interestingly, TrmD proteins from Escherichia coli and Mycobacterium tuberculosis, organisms known to synthesize 3′,5′-cAMP, did not bind this signaling nucleotide. Comparative bioinformatics, mutagenesis, and biochemical analyses revealed that the highly conserved Tyr-86 residue in E. coli TrmD is essential to discriminate between 3′,5′-cAMP and the native substrate AdoMet. Combined with a phylogenetic analysis, these results suggest that amino acids in the substrate binding pocket of TrmD underwent an adaptive evolution to accommodate the emergence of adenylate cyclases and thus the signaling molecule 3′,5′-cAMP. Altogether this further indicates that S. aureus does not produce 3′,5′-cAMP, which would otherwise competitively inhibit an essential enzyme.

Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

“…Despite the known issues ( 40 ), DRaCALA remains one of the most powerful tools devised so far for identifying small ligand binding proteins in a systematic manner. Previously, novel targets of bacterial signaling nucleotides, such as c-di-GMP (cyclic diguanylate monophosphate), c-di-AMP, cAMP, and, recently, also (p)ppGpp (in Staphylococcus aureus ), had successfully been identified by DRaCALA ( 37 – 41 ). To employ DRaCALA, we used the ASKA plasmid library consisting of a complete set of E. coli K-12 genes encoding N-terminally His-tagged proteins encoded by a high-copy-number plasmid ( 42 ).…”

The archaeal triphosphate tunnel metalloenzymeSaTTM defines structural determinants for the diverse activities in the CYTH protein family

“…In cluster 5, unpublished structures annotated before as adenylyl cyclase from Bacillus anthracis and B. halodurans can be found. A member of this cluster from Staphylococcus aureus has further been described to be incompetent in cAMP formation and to be distinct from class IV adenylyl cylases (19).Cluster 3 contains orthologs of mostly archaeal origin from Pyrococcus furiosus and P. horikoshii, as well as Saci_0718 (Uniprot: Q4JAT2). The SSN analysis clearly indicating that the archaeal CYTH enzymes are functionally diverse from CyaB-like class IV adenylyl cyclases.…”

Adenylate Cyclases of Trypanosoma brucei, Environmental Sensors and Controllers of Host Innate Immune Response

“…Cyclic AMP (cAMP) is a ubiquitous metabolite produced from ATP by adenylate cyclase (AC). This molecule is involved in regulation of enzyme activities and/or gene expression in all organisms except in bacteria of the Firmicutes group [ 1 ]. Although this paradigmatic signaling molecule is involved in numerous and varied physiological processes ranging from carbon catabolite repression in bacteria [ 2 ] to chemotaxis mediation in Dictyostelium [ 3 ] and the action of hormones in superior eukaryotes, the downstream effectors of the cAMP pathway and their biological functions still represent an open issue that needs to be clarified [ 4 ].…”