title: 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.
authors.affiliation: From the Section of Microbiology and MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.
authors.forename: Yong
authors.identifier: http://orcid.org/0000-0003-2968-0081
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: WELBIO, Avenue Hippocrate 75, 1200 Brussels, Belgium.|de Duve Institute, Université Catholique de Louvain, Avenue Hippocrate 75, 1200 Brussels, Belgium, and.
authors.forename: Rym
authors.initials: R
authors.lastname: Agrebi
authors.affiliation: From the Section of Microbiology and MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Bellows
authors.affiliation: WELBIO, Avenue Hippocrate 75, 1200 Brussels, Belgium.|de Duve Institute, Université Catholique de Louvain, Avenue Hippocrate 75, 1200 Brussels, Belgium, and.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Collet
authors.affiliation: Research Core Unit Metabolomics, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
authors.forename: Volkhard
authors.initials: V
authors.lastname: Kaever
authors.affiliation: From the Section of Microbiology and MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom, a.grundling@imperial.ac.uk.
authors.forename: Angelika
authors.identifier: http://orcid.org/0000-0002-6235-8687
authors.initials: A
authors.lastname: Gründling
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D000242:Cyclic AMP / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D019143:Evolution, Molecular; D006801:Humans; D007700:Kinetics; D008745:Methylation; D008958:Models, Molecular; D010802:Phylogeny; D011487:Protein Conformation; D012436:S-Adenosylmethionine / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity; D012359:tRNA Methyltransferases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D012436:S-Adenosylmethionine; D000242:Cyclic AMP; D012359:tRNA Methyltransferases
keywords: Staphylococcus aureus (S. aureus); cyclic AMP (cAMP); gram-positive bacteria; protein evolution; signaling