Structural Biochemistry of a Vibrio cholerae Dinucleotide Cyclase Reveals Cyclase Activity Regulation by Folates

Abstract

Cyclic dinucleotides are a newly expanded class of second messengers that contribute to the regulation of multiple different pathways in bacterial, eukaryotic, and archaeal cells. The recently identified Vibrio cholerae dinucleotide cyclase (DncV, the gene product of VC0179) can generate three different cyclic dinucleotides and preferentially synthesize a hybrid cyclic-GMP-AMP. Here, we report the crystal structural and functional studies of DncV. We unexpectedly observed a 5-methyltetrahydrofolate diglutamate (5MTHFGLU2) molecule bound in a surface pocket opposite the nucleotide substrate-binding groove of DncV. Subsequent mutagenesis and functional studies showed that the enzymatic activity of DncV is regulated by folate-like molecules, suggesting the existence of a signaling pathway that links folate-like metabolism cofactors to the regulation of cyclic dinucleotide second messenger synthesis. Sequence analysis showed that the residues involved in 5MTHFGLU2 binding are highly conserved in DncV orthologs, implying the presence of this regulation mechanism in a wide variety of bacteria.

Ancient Origin of cGAS-STING Reveals Mechanism of Universal 2′,3′ cGAMP Signaling

“…Vibrio cholerae DncV is a close structural homolog of human cGAS (Kato et al, 2015; Kranzusch et al, 2014; Zhu et al, 2014), and the enzyme active sites of DncV and cGAS are similar enough that a single amino acid substitution converts human cGAS into a DncV-like 3′,3′ cGAMP synthase (Kranzusch et al, 2014). Given that the nv-cGAS sequence is predicted to have similar changes to catalytic pocket amino acids important for substrate positioning (Figure S2), we hypothesized that the 3′,3′ CDN nv-cGAS product is most likely to be 3′,3′ cGAMP.…”

Versatile modes of cellular regulation via cyclic dinucleotides

“…Reverse engineering of the human cGAS’ active site based on that of DncV produced cGAS variants that synthesized exclusively 3′,3′-cGAMP 32 . Furthermore, a recent study also identified folate as an unexpected regulator of DncV 33 . Although the exact purpose of regulation by folate is not well understood, it is intriguing to note that folate binds to DncV in a similar pocket as double-stranded DNA does to cGAS 33 .…”

“…Furthermore, a recent study also identified folate as an unexpected regulator of DncV 33 . Although the exact purpose of regulation by folate is not well understood, it is intriguing to note that folate binds to DncV in a similar pocket as double-stranded DNA does to cGAS 33 . Together, these observations paint an evolutionary picture in which metazoan cells could have adopted a bacterial cyclase to create, with relatively modest changes, a cytosolic DNA sensor as defence against intracellular pathogens.…”

cGAS and CD-NTase enzymes: structure, mechanism, and evolution

“…In contrast, 'Type II' CD-NTases like Vibrio DncV and many bacterial CD-NTases are catalytically active in the absence of ligand and constitutively synthesize nucleotide signals in vitro [16 ,17 ,18 ,19 ]. Vibrio DncV is inhibited upon binding to folate-like compounds including 5-methyltetrahydrofolic acid [18 ], a metabolite required for nucleotide biosynthesis, suggesting that regulation of Type II enzymes can occur through ligand binding events that repress enzyme function ( Figure 1b). Bacterial CD-NTase operons are frequently encoded within defense islands and have been hypothesized to be involved in phage resistance [19 ,20 ].…”