Structure-guided reprogramming of human cGAS dinucleotide linkage specificity.

Cyclic dinucleotides (CDNs) play central roles in bacterial pathogenesis and innate immunity. The mammalian enzyme cGAS synthesizes a unique cyclic dinucleotide (cGAMP) containing a 2'-5' phosphodiester linkage essential for optimal immune stimulation, but the molecular basis for linkage specificity is unknown. Here, we show that the Vibrio cholerae pathogenicity factor DncV is a prokaryotic cGAS-like enzyme whose activity provides a mechanistic rationale for the unique ability of cGAS to produce 2'-5' cGAMP. Three high-resolution crystal structures show that DncV and human cGAS generate CDNs in sequential reactions that proceed in opposing directions. We explain 2' and 3' linkage specificity and test this model by reprogramming the human cGAS active site to produce 3'-5' cGAMP, leading to selective stimulation of alternative STING adaptor alleles in cells. These results demonstrate mechanistic homology between bacterial signaling and mammalian innate immunity and explain how active site configuration controls linkage chemistry for pathway-specific signaling.

Proteases influence colony aggregation behavior in Vibrio cholerae.

Aggregation behavior provides bacteria protection from harsh environments and threats to survival. Two uncharacterized proteases, LapX and Lap, are important for Vibrio cholerae liquid-based aggregation. Here, we determined that LapX is a serine protease with a preference for cleavage after glutamate and glutamine residues in the P1 position, which processes a physiologically based peptide substrate with a catalytic efficiency of 180 ± 80 M-1s-1. The activity with a LapX substrate identified by a multiplex substrate profiling by mass spectrometry screen was 590 ± 20 M-1s-1. Lap shares high sequence identity with an aminopeptidase (termed VpAP) from Vibrio proteolyticus and contains an inhibitory bacterial prepeptidase C-terminal domain that, when eliminated, increases catalytic efficiency on leucine p-nitroanilide nearly four-fold from 5.4 ± 4.1 × 104 M-1s-1 to 20.3 ± 4.3 × 104 M-1s-1. We demonstrate that LapX processes Lap to its mature form and thus amplifies Lap activity. The increase is approximately eighteen-fold for full-length Lap (95.7 ± 5.6 × 104 M-1s-1) and six-fold for Lap lacking the prepeptidase C-terminal domain (11.3 ± 1.9 × 105 M-1s-1). In addition, substrate profiling reveals preferences for these two proteases that could inform in vivo function. Furthermore, purified LapX and Lap restore the timing of the V. cholerae aggregation program to a mutant lacking the lapX and lap genes. Both proteases must be present to restore WT timing, and thus they appear to act sequentially: LapX acts on Lap, and Lap acts on the substrate involved in aggregation.

A pGpG-specific phosphodiesterase regulates cyclic di-GMP signaling in Vibrio cholerae.

The bacterial second messenger bis-(3'-5')-cyclic diguanylate monophosphate (c-di-GMP) controls various cellular processes, including motility, toxin production, and biofilm formation. c-di-GMP is enzymatically synthesized by GGDEF domain-containing diguanylate cyclases and degraded by HD-GYP domain-containing phosphodiesterases (PDEs) to 2 GMP or by EAL domain-containing PDE-As to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG). Since excess pGpG feedback inhibits PDE-A activity and thereby can lead to the uncontrolled accumulation of c-di-GMP, a PDE that degrades pGpG to 2 GMP (PDE-B) has been presumed to exist. To date, the only enzyme known to hydrolyze pGpG is oligoribonuclease Orn, which degrades all kinds of oligoribonucleotides. Here, we identified a pGpG-specific PDE, which we named PggH, using biochemical approaches in the gram-negative bacteria Vibrio cholerae. Biochemical experiments revealed that PggH exhibited specific PDE activity only toward pGpG, thus differing from the previously reported Orn. Furthermore, the high-resolution structure of PggH revealed the basis for its PDE activity and narrow substrate specificity. Finally, we propose that PggH could modulate the activities of PDE-As and the intracellular concentration of c-di-GMP, resulting in phenotypic changes including in biofilm formation.

Study of the DnaB:DciA interplay reveals insights into the primary mode of loading of the bacterial replicative helicase.

Replicative helicases are essential proteins that unwind DNA in front of replication forks. Their loading depends on accessory proteins and in bacteria, DnaC and DnaI are well characterized loaders. However, most bacteria do not express either of these two proteins. Instead, they are proposed to rely on DciA, an ancestral protein unrelated to DnaC/I. While the DciA structure from Vibrio cholerae shares no homology with DnaC, it reveals similarities with DnaA and DnaX, two proteins involved during replication initiation. As other bacterial replicative helicases, VcDnaB adopts a toroid-shaped homo-hexameric structure, but with a slightly open dynamic conformation in the free state. We show that VcDnaB can load itself on DNA in vitro and that VcDciA stimulates this function, resulting in an increased DNA unwinding. VcDciA interacts with VcDnaB with a 3/6 stoichiometry and we show that a determinant residue, which discriminates DciA- and DnaC/I-helicases, is critical in vivo. Our work is the first step toward the understanding of the ancestral mode of loading of bacterial replicative helicases on DNA. It sheds light on the strategy employed by phage helicase loaders to hijack bacterial replicative helicases and may explain the recurrent domestication of dnaC/I through evolution in bacteria.

A phage satellite tunes inducing phage gene expression using a domesticated endonuclease to balance inhibition and virion hijacking.

Bacteria persist under constant threat of predation by bacterial viruses (phages). Bacteria-phage conflicts result in evolutionary arms races often driven by mobile genetic elements (MGEs). One such MGE, a phage satellite in Vibrio cholerae called PLE, provides specific and robust defense against a pervasive lytic phage, ICP1. The interplay between PLE and ICP1 has revealed strategies for molecular parasitism allowing PLE to hijack ICP1 processes in order to mobilize. Here, we describe the mechanism of PLE-mediated transcriptional manipulation of ICP1 structural gene transcription. PLE encodes a novel DNA binding protein, CapR, that represses ICP1's capsid morphogenesis operon. Although CapR is sufficient for the degree of capsid repression achieved by PLE, its activity does not hinder the ICP1 lifecycle. We explore the consequences of repression of this operon, demonstrating that more stringent repression achieved through CRISPRi restricts both ICP1 and PLE. We also discover that PLE transduces in modified ICP1-like particles. Examination of CapR homologs led to the identification of a suite of ICP1-encoded homing endonucleases, providing a putative origin for the satellite-encoded repressor. This work unveils a facet of the delicate balance of satellite-mediated inhibition aimed at blocking phage production while successfully mobilizing in a phage-derived particle.

ChiS is a noncanonical DNA-binding hybrid sensor kinase that directly regulates the chitin utilization program in Vibrio cholerae.

Two-component signal transduction systems (TCSs) represent a major mechanism that bacteria use to sense and respond to their environment. Prototypical TCSs are composed of a membrane-embedded histidine kinase, which senses an environmental stimulus and subsequently phosphorylates a cognate partner protein called a response regulator that regulates gene expression in a phosphorylation-dependent manner. Vibrio cholerae uses the hybrid histidine kinase ChiS to activate the expression of the chitin utilization program, which is critical for the survival of this facultative pathogen in its aquatic reservoir. A cognate response regulator for ChiS has not been identified and the mechanism of ChiS-dependent signal transduction remains unclear. Here, we show that ChiS is a noncanonical membrane-embedded one-component system that can both sense chitin and directly regulate gene expression via a cryptic DNA binding domain. Unlike prototypical TCSs, we find that ChiS DNA binding is diminished, rather than stimulated, by phosphorylation. Finally, we provide evidence that ChiS likely activates gene expression by directly recruiting RNA polymerase. This work addresses the mechanism of action for a major transcription factor in V. cholerae and highlights the versatility of signal transduction systems in bacterial species.

Structural basis of peptidoglycan endopeptidase regulation.

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over; the latter process is mediated by PG cleavage enzymes, for example, the endopeptidases (EPs). EPs themselves are essential for growth but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., ß-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae Our data suggest that ShyA assumes two drastically different conformations: a more open form that allows for substrate binding and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain 1 from the M23 active site, likely through conformational rearrangement in vivo.

Molecular dynamics modeling of the Vibrio cholera Na+-translocating NADH:quinone oxidoreductase NqrB-NqrD subunit interface.

The Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) is the major Na+ pump in aerobic pathogens such as Vibrio cholerae. The interface between two of the NQR subunits, NqrB and NqrD, has been proposed to harbor a binding site for inhibitors of Na+-NQR. While the mechanisms underlying Na+-NQR function and inhibition remain underinvestigated, their clarification would facilitate the design of compounds suitable for clinical use against pathogens containing Na+-NQR. An in silico model of the NqrB-D interface suitable for use in molecular dynamics simulations was successfully constructed. A combination of algorithmic and manual methods was used to reconstruct portions of the two subunits unresolved in the published crystal structure and validate the resulting structure. Hardware and software optimizations that improved the efficiency of the simulation were considered and tested. The geometry of the reconstructed complex compared favorably to the published V. cholerae Na+-NQR crystal structure. Results from one 1 µs, three 150 ns and two 50 ns molecular dynamics simulations illustrated the stability of the system and defined the limitations of this model. When placed in a lipid bilayer under periodic boundary conditions, the reconstructed complex was completely stable for at least 1 µs. However, the NqrB-D interface underwent a non-physiological transition after 350 ns.

Structural biochemistry of a Vibrio cholerae dinucleotide cyclase reveals cyclase activity regulation by folates.

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.

Inhibition studies of bacterial α-carbonic anhydrases with phenols.

The α-class carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogens Neisseria gonorrhoeae (NgCAα) and Vibrio cholerae (VchCAα) were investigated for their inhibition by a panel of phenols and phenolic acids. Mono-, di- and tri-substituted phenols incorporating additional hydroxyl/hydroxymethyl, amino, acetamido, carboxyl, halogeno and carboxyethenyl moieties were included in the study. The best NgCAα inhibitrs were phenol, 3-aminophenol, 4-hydroxy-benzylalcohol, 3-amino-4-chlorophenol and paracetamol, with KI values of 0.6-1.7 µM. The most effective VchCAα inhibitrs were phenol, 3-amino-4-chlorophenol and 4-hydroxy-benzyl-alcohol, with KI values of 0.7-1.2 µM. Small changes in the phenol scaffold led to drastic effects on the bacterial CA inhibitory activity. This class of underinvestigated bacterial CA inhibitors may thus lead to effective compounds for fighting drug resistant bacteria.

Coumarins effectively inhibit bacterial α-carbonic anhydrases.

Coumarins are known to act as prodrug inhibitors of mammalian α-carbonic anhydrases (CAs, EC 4.2.1.1) but they were not yet investigated for the inhibition of bacterial α-CAs. Here we demonstrate that such enzymes from the bacterial pathogens Neisseria gonorrhoeae (NgCAα) and Vibrio cholerae (VchCAα) are inhibited by a panel of simple coumarins incorporating hydroxyl, amino, ketone or carboxylic acid ester moieties in various positions of the ring system. The nature and the position of the substituents in the coumarin ring were the factors which strongly influenced inhibitory efficacy. NgCAα was inhibited with KIs in the range of 28.6-469.5 µM, whereas VchCAα with KIs in the range of 39.8-438.7 µM. The two human (h)CA isoforms included for comparison reason in the study, hCA I and II, were less prone to inhibition by these compounds, with KIs of 137-948.9 µM for hCA I and of 296.5-961.2 µM for hCA II, respectively. These findings are relevant for discovering coumarin bacterial CA inhibitors with selectivity for the bacterial over human isoform, with potential applications as novel antibacterial agents.

Vibrio cholerae FeoB contains a dual nucleotide-specific NTPase domain essential for ferrous iron uptake.

The Feo ferrous iron transporter is widely distributed among bacteria and archaea, but its mechanism of transport has not been fully elucidated. In Vibrio cholerae, the transport system requires three proteins: the small cytosolic proteins FeoA and FeoC and a large cytoplasmic-membrane-associated protein FeoB, which has an N-terminal G-protein domain. We show that, in contrast to Escherichia coli FeoB, which is solely a GTPase, the V. cholerae and Helicobacter pylori FeoB proteins have both GTPase and ATPase activity. In V. cholerae, mutation of the G4 motif, responsible for hydrogen bonding with the guanine base, abolished the GTPase activity but not ATPase activity. The ATPase activity of the G4 motif mutants was sufficient for Feo function in the absence of GTPase. We show that the serine and asparagine residues in the G5 motif likely play a role in the ATPase activity, and substitution of these residues with those found in the corresponding positions in E. coli FeoB resulted in similar nucleotide hydrolysis activity in the E. coli protein. These results add significantly to our understanding of the NTPase domain of FeoB and its role in Feo function.

HutW from Vibrio cholerae Is an Anaerobic Heme-Degrading Enzyme with Unique Functional Properties.

Increasing antibiotic resistance, and a growing recognition of the importance of the human microbiome, demand that new therapeutic targets be identified. Characterization of metabolic pathways that are unique to enteric pathogens represents a promising approach. Iron is often the rate-limiting factor for growth, and Vibrio cholerae, the causative agent of cholera, has been shown to contain numerous genes that function in the acquisition of iron from the environment. Included in this arsenal of genes are operons dedicated to obtaining iron from heme and heme-containing proteins. Given the persistence of cholera, an important outstanding question is whether V. cholerae is capable of anaerobic heme degradation as was recently reported for enterohemorrhagic Escherichia coli O157:H7. In this work, we demonstrate that HutW from V. cholerae is a radical S-adenosylmethionine methyl transferase involved in the anaerobic opening of the porphyrin ring of heme. However, in contrast to the enzyme ChuW, found in enterohemorrhagic E. coli O157:H7, there are notable differences in the mechanism and products of the HutW reaction. Of particular interest are data that demonstrate HutW will catalyze ring opening as well as tetrapyrrole reduction and can utilize reduced nicotinamide adenine dinucleotide phosphate as an electron source. The biochemical and biophysical properties of HutW are presented, and the evolutionary implications are discussed.

Enzymatic Synthesis of 3'-5', 3'-5' Cyclic Dinucleotides, Their Binding Properties to the Stimulator of Interferon Genes Adaptor Protein, and Structure/Activity Correlations.

The 3'-5', 3'-5' cyclic dinucleotides (3'3'CDNs) are bacterial second messengers that can also bind to the stimulator of interferon genes (STING) adaptor protein in vertebrates and activate the host innate immunity. Here, we profiled the substrate specificity of four bacterial dinucleotide synthases from Vibrio cholerae (DncV), Bacillus thuringiensis (btDisA), Escherichia coli (dgcZ), and Thermotoga maritima (tDGC) using a library of 33 nucleoside-5'-triphosphate analogues and then employed these enzymes to synthesize 24 3'3'CDNs. The STING affinity of CDNs was evaluated in cell-based and biochemical assays, and their ability to induce cytokines was determined by employing human peripheral blood mononuclear cells. Interestingly, the prepared heterodimeric 3'3'CDNs bound to the STING much better than their homodimeric counterparts and showed similar or better potency than bacterial 3'3'CDNs. We also rationalized the experimental findings by in-depth STING-CDN structure-activity correlations by dissecting computed interaction free energies into a set of well-defined and intuitive terms. To this aim, we employed state-of-the-art methods of computational chemistry, such as quantum mechanics/molecular mechanics (QM/MM) calculations, and complemented the computed results with the {STING:3'3'c-di-ara-AMP} X-ray crystallographic structure. QM/MM identified three outliers (mostly homodimers) for which we have no clear explanation of their impaired binding with respect to their heterodimeric counterparts, whereas the R2 = 0.7 correlation between the computed ΔG'int_rel and experimental ΔTm's for the remaining ligands has been very encouraging.

Electrostatics and water occlusion regulate covalently-bound flavin mononucleotide cofactors of Vibrio cholerae respiratory complex NQR.

Proteins like NADH:ubiquinone oxidoreductase (NQR), an essential enzyme and ion pump in the physiology of several pathogenic bacteria, tightly regulate the redox properties of their cofactors. Although flavin mononucleotide (FMN) is fully reduced in aqueous solution, FMN in subunits B and C of NQR exclusively undergo one-electron transitions during its catalytic cycle. Here, we perform ab initio calculations and molecular dynamics simulations to elucidate the mechanisms that regulate the redox state of FMN in NQR. QM/MM calculations show that binding site electrostatics disfavor anionic forms of FMNH2 , but permit a neutral form of the fully reduced flavin. The potential energy surface is unaffected by covalent bonding between FMN and threonine. Molecular dynamics simulations show that the FMN binding sites are inaccessible by water, suggesting that further reductions of the cofactors are limited or prohibited by the availability of water and other proton donors. These findings provide a deeper understanding of the mechanisms used by NQR to regulate electron transfer through the cofactors and perform its physiologic role. They also provide the first, to our knowledge, evidence of the simple concept that proteins regulate flavin redox states via water occlusion.

Cryo-EM structure of Vibrio cholerae aldehyde-alcohol dehydrogenase spirosomes.

Aldehyde-alcohol dehydrogenase (AdhE) is a metabolic enzyme and virulence factor in bacteria. E. coli AdhE (eAdhE) multimerizes into spirosomes that are essential for enzymatic activity. However, it is unknown whether AdhE structure is conserved in divergent bacteria. Here, we present the cryo-EM structure of AdhE (vAdhE) from Vibrio cholerae to 4.31 Å resolution. Overall, vAdhE spirosomes are similar to eAdhE with conserved subunit arrangement. However, divergences in key oligomerization residues cause vAdhE to form labile spirosomes with lower enzymatic activity. Mutating the vAdhE oligomerization interface to mimic eAdhE increases spirosome stability and enzymatic activity to levels comparable to eAdhE. These results support the generality of AdhE spirosome structures, and provide a structural basis to target vAdhE to attenuate bacterial virulence.

Inhibition of α-, ß- and γ-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae with aromatic sulphonamides and clinically licenced drugs - a joint docking/molecular dynamics study.

The binding mode of aromatic sulphonamides and clinically licenced drugs to the three carbonic anhydrase (CA, EC 4.2.1.1) isoforms from the human pathogen V. cholerae was here thouroghly characterised by a joint docking and molecular dynamics in silico protocol. In fact, VchCA, VchCAß, and VchCAγ are crucial in the pathogen life cycle and growth and represent innovative targets to fight V. cholerae proliferation overcoming the spreading chemoresistance to the available drugs. A set of 40 sulphonamides/sulfamates VchCAs inhibitors was studied using the proteins homology built 3 D models unveiling the key and stable interactions responsible for a potent CA inhibition. This study has the aim to offer insights and guidelines for the future rational design of potent and selective inhibitors targeting CA isoforms from V. cholerae or other human pathogens.

Direct activation of a phospholipase by cyclic GMP-AMP in El Tor Vibrio cholerae.

Sensing and responding to environmental changes is essential for bacteria to adapt and thrive, and nucleotide-derived second messengers are central signaling systems in this process. The most recently identified bacterial cyclic dinucleotide second messenger, 3', 3'-cyclic GMP-AMP (cGAMP), was first discovered in the El Tor biotype of Vibrio cholerae The cGAMP synthase, DncV, is encoded on the VSP-1 pathogenicity island, which is found in all El Tor isolates that are responsible for the current seventh pandemic of cholera but not in the classical biotype. We determined that unregulated production of DncV inhibits growth in El Tor V. cholerae but has no effect on the classical biotype. This cGAMP-dependent phenotype can be suppressed by null mutations in vc0178 immediately 5' of dncV in VSP-1. VC0178 [renamed as cGAMP-activated phospholipase in Vibrio (CapV)] is predicted to be a patatin-like phospholipase, and coexpression of capV and dncV is sufficient to induce growth inhibition in classical V. cholerae and Escherichia coli Furthermore, cGAMP binds to CapV and directly activates its hydrolase activity in vitro. CapV activated by cGAMP in vivo degrades phospholipids in the cell membrane, releasing 16:1 and 18:1 free fatty acids. Together, we demonstrate that cGAMP activates CapV phospholipase activity to target the cell membrane and suggest that acquisition of this second messenger signaling pathway may contribute to the emergence of the El Tor biotype as the etiological agent behind the seventh cholera pandemic.

Impact of Na+-Translocating NADH:Quinone Oxidoreductase on Iron Uptake and nqrM Expression in Vibrio cholerae.

The Na+ ion-translocating NADH:quinone oxidoreductase (NQR) from Vibrio cholerae is a membrane-bound respiratory enzyme which harbors flavins and Fe-S clusters as redox centers. The NQR is the main producer of the sodium motive force (SMF) and drives energy-dissipating processes such as flagellar rotation, substrate uptake, ATP synthesis, and cation-proton antiport. The NQR requires for its maturation, in addition to the six structural genes nqrABCDEF, a flavin attachment gene, apbE, and the nqrM gene, presumably encoding a Fe delivery protein. We here describe growth studies and quantitative real-time PCR for the V. cholerae O395N1 wild-type (wt) strain and its mutant Δnqr and ΔubiC strains, impaired in respiration. In a comparative proteome analysis, FeoB, the membrane subunit of the uptake system for Fe2+ (Feo), was increased in V. choleraeΔnqr In this study, the upregulation was confirmed on the mRNA level and resulted in improved growth rates of V. choleraeΔnqr with Fe2+ as an iron source. We studied the expression of feoB on other respiratory enzyme deletion mutants such as the ΔubiC mutant to determine whether iron transport is specific to the absence of NQR resulting from impaired respiration. We show that the nqr operon comprises, in addition to the structural nqrABCDEF genes, the downstream apbE and nqrM genes on the same operon and demonstrate induction of the nqr operon by iron in V. cholerae wt. In contrast, expression of the nqrM gene in V. choleraeΔnqr is repressed by iron. The lack of functional NQR has a strong impact on iron homeostasis in V. cholerae and demonstrates that central respiratory metabolism is interwoven with iron uptake and regulation.IMPORTANCE Investigating strategies of iron acquisition, storage, and delivery in Vibrio cholerae is a prerequisite to understand how this pathogen thrives in hostile, iron-limited environments such as the human host. In addition to highlighting the maturation of the respiratory complex NQR, this study points out the influence of NQR on iron metabolism, thereby making it a potential drug target for antibiotics.

HD-[HD-GYP] Phosphodiesterases: Activities and Evolutionary Diversification within the HD-GYP Family.

Cyclic dinucleotides are signaling molecules that modulate many processes, including immune response and virulence factor production. Their cellular levels in bacteria are fine-tuned by metal-dependent phosphodiesterases, namely, the EAL and HD-GYP proteins, with HD-GYPs belonging to the larger HD domain superfamily. In this study, we first focus on the catalytic properties and the range of metal ions and substrates of the HD-[HD-GYP] subfamily, consisting of two HD domains. We identified SO3491 as a homologue of VCA0681 and the second example of an HD-[HD-GYP]. Both proteins hydrolyze c-di-GMP and 3'3'c-GAMP and coordinate various metal ions, but only Fe and to a lesser extent Co support hydrolysis. The proteins are active only in the diferrous form and not in the one-electron more oxidized FeIIFeIII state. Although the C-terminal HD-GYP domain is essential for activity, the role of the N-terminal HD domain remains unknown. We show that the N-terminal site is important for protein stability, influences the individual apparent kcat and KM (but not kcat/KM), and cannot bind c-di-GMP, thus precluding its involvement in cyclic dinucleotide sensing. We proceeded to perform phylogenetic analyses to examine the distribution and functional relationships of the HD-[HD-GYP]s to the rest of the HD-GYPs. The phylogeny provides a correlation map that draws a link between the evolutionary and functional diversification of HD-GYPs, serving as a template for predicting the chemical nature of the metallocofactor, level of activity, and reaction outcome.