HD-[HD-GYP] Phosphodiesterases: Activities and Evolutionary Diversification within the HD-GYP Family.
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ABSTRACT:
SUBMITTER: Sun S
PROVIDER: S-EPMC8177754 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:3',3'-cyclic GMP-AMP (cGAMP) is the third cyclic dinucleotide (CDN) to be discovered in bacteria. No activators of cGAMP signaling have yet been identified, and the signaling pathways for cGAMP have been inferred to display a narrow distribution based upon the characterized synthases, DncV and Hypr GGDEFs. Here, we report that the ubiquitous second messenger cyclic AMP (cAMP) is an activator of the Hypr GGDEF enzyme GacB from Myxococcus xanthus. Furthermore, we show that GacB is inhibited directly by cyclic di-GMP, which provides evidence for cross-regulation between different CDN pathways. Finally, we reveal that the HD-GYP enzyme PmxA is a cGAMP-specific phosphodiesterase (GAP) that promotes resistance to osmotic stress in M. xanthus. A signature amino acid change in PmxA was found to reprogram substrate specificity and was applied to predict the presence of non-canonical HD-GYP phosphodiesterases in many bacterial species, including phyla previously not known to utilize cGAMP signaling.
| S-EPMC7209772 | biostudies-literature
Project description:In biofilms, the bacterial community optimizes the strategies to sense the environment and to communicate from cell to cell. A key player in the development of a bacterial biofilm is the second messenger c-di-GMP, whose intracellular levels are modulated by the opposite activity of diguanylate cyclases and phosphodiesterases. Given the huge impact of bacterial biofilms on human health, understanding the molecular details of c-di-GMP metabolism represents a critical step in the development of novel therapeutic approaches against biofilms. In this study, we present a detailed biochemical characterization of two c-di-GMP phosphodiesterases of the HD-GYP subtype from the human pathogen Pseudomonas aeruginosa, namely PA4781 and PA4108. Upstream of the catalytic HD-GYP domain, PA4781 contains a REC domain typical of two-component systems, while PA4108 contains an uncharacterized domain of unknown function. Our findings shed light on the activity and catalytic mechanism of these phosphodiesterases. We show that both enzymes hydrolyse c-di-GMP in a two-step reaction via the linear intermediate pGpG and that they produce GMP in vitro at a surprisingly low rate. In addition, our data indicate that the non-phosphorylated REC domain of PA4781 prevents accessibility of c-di-GMP to the active site. Both PA4108 and phosphorylated PA4781 are also capable to use pGpG as an alternative substrate and to hydrolyse it into GMP; the affinity of PA4781 for pGpG is one order of magnitude higher than that for c-di-GMP. These results suggest that these enzymes may not work (primarily) as genuine phosphodiesterases. Moreover, the unexpected affinity of PA4781 for pGpG may indicate that pGpG could also act as a signal molecule in its own right, thus further widening the c-di-GMP-related signalling scenario.
| S-EPMC3774798 | biostudies-literature
Project description:Cyclic-di-GMP is a near-ubiquitous bacterial second messenger that is important in localized signal transmission during the control of various processes, including virulence and switching between planktonic and biofilm-based lifestyles. Cyclic-di-GMP is synthesized by GGDEF diguanylate cyclases and hydrolyzed by EAL or HD-GYP phosphodiesterases, with each functional domain often appended to distinct sensory modules. HD-GYP domain proteins have resisted structural analysis, but here we present the first structural representative of this family (1.28 Å), obtained using the unusual Bd1817 HD-GYP protein from the predatory bacterium Bdellovibrio bacteriovorus. Bd1817 lacks the active-site tyrosine present in most HD-GYP family members yet remains an excellent model of their features, sharing 48% sequence similarity with the archetype RpfG. The protein structure is highly modular and thus provides a basis for delineating domain boundaries in other stimulus-dependent homologues. Conserved residues in the HD-GYP family cluster around a binuclear metal center, which is observed complexed to a molecule of phosphate, providing information on the mode of hydroxide ion attack on substrate. The fold and active site of the HD-GYP domain are different from those of EAL proteins, and restricted access to the active-site cleft is indicative of a different mode of activity regulation. The region encompassing the GYP motif has a novel conformation and is surface exposed and available for complexation with binding partners, including GGDEF proteins. It is becoming apparent that many bacteria use the signaling molecule cyclic-di-GMP to regulate a variety of processes, most notably, transitions between motility and sessility. Importantly, this regulation is central to several traits implicated in chronic disease (adhesion, biofilm formation, and virulence gene expression). The mechanisms of cyclic-di-GMP synthesis via GGDEF enzymes and hydrolysis via EAL enzymes have been suggested by the analysis of several crystal structures, but no information has been available to date for the unrelated HD-GYP class of hydrolases. Here we present the multidomain structure of an unusual member of the HD-GYP family from the predatory bacterium Bdellovibrio bacteriovorus and detail the features that distinguish it from the wider structural family of general HD fold hydrolases. The structure reveals how a binuclear iron center is formed from several conserved residues and provides a basis for understanding HD-GYP family sequence requirements for c-di-GMP hydrolysis.
| S-EPMC3188283 | biostudies-literature
Project description:UnlabelledThe intracellular level of the bacterial secondary messenger cyclic di-3',5'-GMP (c-di-GMP) is determined by a balance between its biosynthesis and degradation, the latter achieved via dedicated phosphodiesterases (PDEs) bearing a characteristic EAL or HD-GYP domain. We here report the crystal structure of PA4781, one of the three Pseudomonas aeruginosa HD-GYP proteins, which we have previously characterized in vitro. The structure shows a bimetallic active site whose metal binding mode is different from those of both HD-GYP PDEs characterized so far. Purified PA4781 does not contain iron in the active site as for other HD-GYPs, and we show that it binds to a wide range of transition metals with similar affinities. Moreover, the structural features of PA4781 indicate that this is preferentially a pGpG binding protein, as we previously suggested. Our results point out that the structural features of HD-GYPs are more complex than predicted so far and identify the HD-GYP domain as a conserved scaffold which has evolved to preferentially interact with a partner GGDEF but which harbors different functions obtained through diversification of the active site.ImportanceIn bacteria, the capability to form biofilms, responsible for increased pathogenicity and antibiotic resistance, is almost universally stimulated by the second messenger cyclic di-GMP (c-di-GMP). To design successful strategies for targeting biofilm formation, a detailed characterization of the enzymes involved in c-di-GMP metabolism is crucial. We solved the structure of the HD-GYP domain of PA4781 from Pseudomonas aeruginosa, involved in c-di-GMP degradation. This is the third structure of this class of phosphodiesterases to be solved, and with respect to its homologues, it shows significant differences both in the nature and in the binding mode of the coordinated metals, indicating that HD-GYP proteins are able to fine-tune their function, thereby increasing the chances of the microorganism to adapt to different environmental needs.
| S-EPMC4372746 | biostudies-literature
Project description:In this study, we carried out an evolutionary, transcriptional, and functional analyses of the trihelix transcription factor family. A total of 319 trihelix members, identified from 11 land plant species, were classified into five clades. The results of phylogeny indicate the binding domains of GT1 and GT2 diverged early in the existence of land plants. Genomic localization revealed that the trihelix family members were highly conserved among cereal species, even though some homeologs generated during the tetraploidy of maize were lost. Three-dimensional structural analyses and an examination of subcellular localization of this family supported the involvement of all five clades in transcriptional regulation. Furthermore, the family members from all clades in sorghum and rice showed a broad and dynamic expression pattern in response to abiotic stresses, indicating regulatory subfunctionalization of their original functions. This finding is further supported by the phenotypes of enhanced tolerance to cold, salt, and drought in transgenic plants overexpressing Sb06g023980 and Sb06g024110. In contrast, few Arobidopsis genes showed inducible expression under abiotic stress conditions, which may indicate a functional shift. Finally, our co-expression analysis points to the involvement of this family in various metabolic processes, implying their further functional divergence.
| S-EPMC4195496 | biostudies-literature
Project description:In a phylogenetic analysis of vertebrate transferrins (TFs), six major clades (subfamilies) were identified: (a) S, the mammalian serotransferrins; (b) ICA, the mammalian inhibitor of carbonic anhydrase (ICA) homologs; (c) L, the mammalian lactoferrins; (d) O, the ovotransferrins of birds and reptiles; (e) M, the melanotransferrins of bony fishes, amphibians, reptiles, birds, and mammals; and (f) M-like, a newly identified TF subfamily found in bony fishes, amphibians, reptiles, and birds. A phylogenetic tree based on the joint alignment of N-lobes and C-lobes supported the hypothesis that three separate events of internal duplication occurred in vertebrate TFs: (a) in the common ancestor of the M subfamily, (b) in the common ancestor of the M-like subfamily, and (c) in the common ancestor of other vertebrate TFs. The S, ICA, and L subfamilies were found only in placental mammals, and the phylogenetic analysis supported the hypothesis that these three subfamilies arose by gene duplication after the divergence of placental mammals from marsupials. The M-like subfamily was unusual in several respects, including the presence of a uniquely high proportion of clade-specific conserved residues, including distinctive but conserved residues in the sites homologous to those functioning in carbonate binding of human serotransferrin. The M-like family also showed an unusually high proportion of cationic residues in the positively charged region corresponding to human lactoferrampin, suggesting a distinctive role of this region in the M-like subfamily, perhaps in antimicrobial defense.
| S-EPMC4255964 | biostudies-literature
Project description:HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'?5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites.
| S-EPMC5055813 | biostudies-literature
Project description:A protein function is carried out by a specific domain localized at a specific position. In the present study, we report that, within a gene, a specific amino acid sequence can move between a certain position and another position. This was discovered when the sequences of restriction-modification systems within the bacterial species Helicobacter pylori were compared. In the specificity subunit of Type I restriction-modification systems, DNA sequence recognition is mediated by target recognition domain 1 (TRD1) and TRD2. To our surprise, several sequences are shared by TRD1 and TRD2 of genes (alleles) at the same locus (chromosomal location); these domains appear to have moved between the two positions. The gene/protein organization can be represented as x-(TRD1)-y-x-(TRD2)-y, where x and y represent repeat sequences. Movement probably occurs by recombination at these flanking DNA repeats. In accordance with this hypothesis, recombination at these repeats also appears to decrease two TRDs into one TRD or increase these two TRDs to three TRDs (TRD1-TRD2-TRD2) and to allow TRD movement between genes even at different loci. Similar movement of domains between TRD1 and TRD2 was observed for the specificity subunit of a Type IIG restriction enzyme. Similar movement of domain between TRD1 and TRD2 was observed for Type I restriction-modification enzyme specificity genes in two more eubacterial species, Streptococcus pyogenes and Mycoplasma agalactiae. Lateral domain movements within a protein, which we have designated DOMO (domain movement), represent novel routes for the diversification of proteins.
| S-EPMC3077401 | biostudies-other
Project description:Rhesus (Rh) proteins were first identified in human erythroid cells and recently in other tissues. Like ammonia transporter (Amt) proteins, their only homologues, Rh proteins have the 12 transmembrane-spanning segments characteristic of transporters. Many think Rh and Amt proteins transport the same substrate, NH(3)/NH(4)(+), whereas others think that Rh proteins transport CO(2) and Amt proteins NH(3). In the latter view, Rh and Amt are different biological gas channels. To reconstruct the phylogeny of the Rh family and study its coexistence with and relationship to Amt in depth, we analyzed 111 Rh genes and 260 Amt genes. Although Rh and Amt are found together in organisms as diverse as unicellular eukaryotes and sea squirts, Rh genes apparently arose later, because they are rare in prokaryotes. However, Rh genes are prominent in vertebrates, in which Amt genes disappear. In organisms with both types of genes, Rh had apparently diverged away from Amt rapidly and then evolved slowly over a long period. Functionally divergent amino acid sites are clustered in transmembrane segments and around the gas-conducting lumen recently identified in Escherichia coli AmtB, in agreement with Rh proteins having new substrate specificity. Despite gene duplications and mutations, the Rh paralogous groups all have apparently been subject to strong purifying selection indicating functional conservation. Genes encoding the classical Rh proteins in mammalian red cells show higher nucleotide substitution rates at nonsynonymous codon positions than other Rh genes, a finding that suggests a possible role for these proteins in red cell morphogenetic evolution.
| S-EPMC1266151 | biostudies-literature
Project description:The Sm family of proteins is closely associated with RNA metabolism throughout all life. These proteins form homomorphic and heteromorphic rings consisting of six or seven subunits with a characteristic central pore, the presence of which is critical for binding U-rich regions of single-stranded RNA. Eubacteria and Archaea typically carry one or two forms of Sm proteins and assemble one homomorphic ring per Sm protein. Eukaryotes typically carry 16 or more Sm proteins that assemble to form heteromorphic rings which lie at the center of a number of critical RNA-associated small nuclear ribonucleoproteins (snRNPs). High Sm protein diversity and heteromorphic Sm rings are features stretching back to the origin of eukaryotes; very deep phylogenetic divisions among existing Sm proteins indicate simultaneous evolution across essentially all existing eukaryotic life. Two basic forms of heteromorphic Sm rings are found in eukaryotes. Fixed Sm rings are highly stable and static and are assembled around an RNA cofactor. Flexible Sm rings also stabilize and chaperone RNA but assemble in the absence of an RNA substrate and, more significantly, associate with and dissociate from RNA substrates more freely than fixed rings. This suggests that the conformation of flexible Sm rings might be modified in some specific manner to facilitate association and dissociation with RNA. Diversification of eukaryotic Sm proteins may have been initiated by gene transfers and/or genome clashes that accompanied the origin of the eukaryotic cell itself, with further diversification driven by a greater need for steric specificity within increasingly complex snRNPs.
| S-EPMC3888151 | biostudies-literature