title: 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.
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720;
authors.forename: Zachary F
authors.initials: ZF
authors.lastname: Hallberg
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;
authors.forename: Xin C
authors.initials: XC
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720;
authors.forename: Todd A
authors.initials: TA
authors.lastname: Wright
authors.affiliation: Department of Biology, Texas A&M University, College Station, TX 77843.
authors.forename: Beiyan
authors.initials: B
authors.lastname: Nan
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720;
authors.forename: Omer
authors.initials: O
authors.lastname: Ad
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720;
authors.forename: Jongchan
authors.initials: J
authors.lastname: Yeo
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720; Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; mingch@berkeley.edu.
authors.forename: Ming C
authors.initials: MC
authors.lastname: Hammond
mesh_terms: D020564:Deltaproteobacteria / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D009712:Nucleotides, Cyclic / Q000096:biosynthesis*; D019761:Phosphorus-Oxygen Lyases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D029968:Escherichia coli Proteins; D009712:Nucleotides, Cyclic; C584311:cyclic guanosine monophosphate-adenosine monophosphate; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase
keywords: bacterial signaling; cyclic dinucleotides; fluorescent biosensor; second messengers; surface sensing
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