Hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3′, 3′-cGAMP)

Abstract

Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3′, 3′-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling.bacterial signaling | surface sensing | second messengers | cyclic dinucleotides | fluorescent biosensor

A Novel Phosphodiesterase of the GdpP Family Modulates Cyclic di-AMP Levels in Response to Cell Membrane Stress in Daptomycin-Resistant Enterococci

“…Additionally, an unknown cofactor or stress may switch the function of GdpP from c-di-AMP phosphodiesterase (DHH/DHHA1 domain activity) to c-di-AMP synthase (putative GGDEF activity) (39). While we were unable to demonstrate that the E. faecalis GdpP GGDEF domain has c-di-AMP synthase activity, GGDEF domains of other proteins do have demonstrated synthase activity (39). How the PAS domain of GdpP might regulate the known DHH/ DHHA1 domain phosphodiesterase activity, as well as the potential cryptic activities of the GGDEF domain, remains unknown.…”

“…Together, these observations suggest that the E. faecalis GdpP GGDEF domain has additional activities in vivo beyond weak ATP hydrolysis. Recently, the GGDEF domains from Deltaproteobacteria were also reported to have a hybrid dinucleotide cyclase activity and can synthesize c-di-AMP, c-di-GMP, and cyclic AMP-GMP (3=,3=-cGAMP) (39). The functional role of the PAS and GGDEF domains is the subject of ongoing investigation.…”

Progress in Understanding the Molecular Basis Underlying Functional Diversification of Cyclic Dinucleotide Turnover Proteins

“…Accordingly, alteration of a few amino acids can alter the substrate specificity of nucleotides and sugars. Although it is the common perception that cyclic di-GMP synthases can be readily identified in bacterial genomes as being members of the GGDEF domain superfamily, GGDEF domain proteins that predominantly synthesize cyclic GMP-AMP, in parallel with cyclic di-GMP and cyclic di-AMP, have recently been identified (31). The relative specificity of cyclic GMP-AMP synthase activity as opposed to stringently using GTP as the substrate on this specific protein scaffold is determined by the amino acid serine, which has replaced aspartate at position 344 (designation according to PleD sequence), a key contact residue in the base binding pocket.…”

Diguanylate cyclase activity of the Mycobacterium leprae T cell antigen ML1419c

“…Importantly, the environmental cues that are perceived by the PAS sensor domains of DgcA would be expected to alter downstream gene expression and protein function via the activity of DgcA-derived cyclic di-GMP. Previous research groups have overexpressed GGDEF proteins similar to DgcA in order to study protein activity in the absence of activating signal (Kulasakara et al, 2006;Hickman & Harwood, 2008), and it is likely that elevated protein levels facilitate the dimerization and activation of these DGCs (Hallberg et al, 2016). Thus, it is possible that the DGC activity demonstrated by the recombinant expression of M. leprae dgcA in P. aeruginosa was a result of protein abundance or interaction of the DgcA PAS domains with an environmental signal.…”