Discovery of a cAMP deaminase that quenches cyclic AMP-dependent regulation

The amidohydrolase (AHS) enzyme superfamily is a well-characterized group of enzymes first identified by Holm and Sander based on the similarities in the three-dimensional structures of phosphotriesterase, adenosine deaminase and urease 1. The proteins of the AHS family fold into a distorted (β/α)8-barrel with conserved metal binding residues at the C-terminal ends of β-strands 1, 4, 5, 6 and 8. These enzymes have either mononuclear or binuclear metal centers which activate a hydrolytic water for nucleophilic attack and are involved in the metabolism of amino acids, sugars, nucleic acids, and organophosphate esters 2. NCBI has divided the AHS into 24 clusters of orthologous groups (COGs) and annotated all bacterial proteins in COG1816 as enzymes that catalyze the deamination of adenosine. The structure and mechanism of action of adenosine deaminases from several organisms have been studied and the sequence similarity network3, for COG1816 is given in Figure 1. Enzymes experimentally verified as adenosine deaminases are found in Group 5, which contains the Escherichia coli K-12 adenosine deaminase (locus tag: b1623). Additional adenosine deaminases are found in Groups 1, 2, 6, and 8. The essential residues for adenosine deaminase activity 2 include an HxHxD motif following β-strand 1, an aspartate following β-strand 3, a glycine following β-strand 4, an HxxE motif following β-strand 5, a histidine following β-strand 6, and a pair of aspartates following β-strand 8 2. Additional functions found in COG1816 include adenine deaminase and cytokinin deaminase in Group 3. Despite an overall amino acid sequence identity of ~30% to E. coli adenosine deaminase, the proteins of Group 18 cannot deaminate adenosine. We have identified a protein of Group 18 of COG1816 that specifically deaminates the signaling molecule cAMP to cyclic-3’, 5’-inosine monophosphate (cIMP) and lacks activity on adenosine. We have named this enzyme CadD for cyclic adenylate deaminase. Figure 1 Sequence similarity network created using Cytoscape (www.cytoscape.org) of COG1816 from the amidohydrolase superfamily. Each node in the network represents a single sequence, and each edge (depicted as lines) represents the pairwise connection between ... The universal signaling molecule cAMP is found in all three domains of life where it plays key roles as a secondary messenger that transmits signals to regulate physiological processes such as sugar and lipid metabolism, cardiac function, cell growth and differentiation 4:Antoni, 2012 #11, 5. Action of cAMP is mediated by binding to a variety of cAMP receptor proteins. These receptor proteins are divided into four main classes: cyclic nucleotide-dependent kinases, cyclic nucleotide-gated ion channels, guanine nucleotide exchange factors and bacterial cAMP-dependent transcription factors (Crp) 4. In the case of Crp proteins cAMP binding results in a large well-understood allosteric transition that allows binding to specific DNA sequences 6–8. The deamination product of cAMP, cIMP, has been reported as a compound released into the growth medium by cultures of Corynebacterium murisepticum and Microbacterium sp. 205 9, 10 and a component of mammalian tissue culture cells 11, although in each of these systems the means of cIMP production are unknown. Deamination of cAMP by a broad specificity fungal adenosine deaminase 12 and in toad bladder extracts 13 has been reported. This work describes the discovery and in vivo function of a cAMP deaminase from the human pathogen, Leptospira interrogans 14, a bacterium transmitted by zoonotic contact that can cause potentially fatal diseases such as renal failure and pulmonary hemorrhage 15.