An HD-GYP Cyclic Di-Guanosine Monophosphate Phosphodiesterase with a Non-Heme Diiron–Carboxylate Active Site

Miner, Kyle D.; Klose, Karl E.; Kurtz, Donald M.

doi:10.1021/bi4009215

Cited by 22 publications

(56 citation statements)

References 7 publications

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“…Strikingly, the structure shows a bimetallic active site whose metal binding mode is different from those of both Bd1817 and PmGH. Purified PA4781 did not contain iron in the active site as for previously reported HD-GYPs (14,16,18), and we show that it binds to a wide range of transition metals with similar affinities. Moreover, the structural features of PA4781 are in agreement with our previous data, indicating that this is preferentially a pGpG binding protein (15).…”

supporting

confidence: 69%

Structural Basis of Functional Diversification of the HD-GYP Domain Revealed by the Pseudomonas aeruginosa PA4781 Protein, Which Displays an Unselective Bimetallic Binding Site

et al. 2015

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The 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. The dinucleotide cyclic di-GMP (c-di-GMP) serves as a core signal in the coordinated lifestyle switch of most bacteria in response to different environmental stimuli. In particular, high levels of c-di-GMP generally flag hostile conditions and result in reduced motility, attachment, and biofilm formation, while low levels of c-di-GMP indicate that it is time to go back to virulence and cell division (1-5). The intracellular levels of c-di-GMP are fine-tuned by the opposite action of dedicated enzymes: diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) (6). The first class, characterized by a GGDEF domain, synthesize c-di-GMP from two GTP molecules, while the second one hydrolyzes it. PDEs are further divided into two unrelated superfamilies of hydrolases: the more abundant EAL proteins, which degrade c-di-GMP to the linear dinucleotide pGpG, and the HD-GYP proteins, which are able to hydrolyze c-di-GMP into two molecules of GMP. Bacteria display a wide variety of genes encoding these three enzymatic domains,...

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supporting

confidence: 69%

Structural Basis of Functional Diversification of the HD-GYP Domain Revealed by the Pseudomonas aeruginosa PA4781 Protein, Which Displays an Unselective Bimetallic Binding Site

et al. 2015

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“…Detailed kinetic analyses indicate that PA4781 has low enzymatic activity but hydrolyzes 5=-pGpG more effectively than cyclic di-GMP (81). Although similar kinetic experiments on other HD-GYP domain proteins have not been reported, the available evidence suggests that differences in the relative activity against 5=-pGpG compared to cyclic di-GMP do occur (66,69,71,76,77,83,84).…”

Section: Diversity In Substrate Binding and Catalysismentioning

confidence: 72%

“…The activity of VCA0681 requires Fe(II) at the bimetallic center, and derivatives with Fe(III) are inactive, suggesting that the activity of this protein is redox regulated (76). Also, isolated TM0186 from Thermotoga maritima with two Fe(III) atoms is inactive; reduction to Fe(II) enables the enzyme to generate 5=-pGpG but not GMP.…”

Section: Diversity In Metal Bindingmentioning

confidence: 99%

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

2017

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Cyclic di-GMP was the first cyclic dinucleotide second messenger described, presaging the discovery of additional cyclic dinucleotide messengers in bacteria and eukaryotes. The GGDEF diguanylate cyclase (DGC) and EAL and HD-GYP phosphodiesterase (PDE) domains conduct the turnover of cyclic di-GMP. These three unrelated domains belong to superfamilies that exhibit significant variations in function, and they include both enzymatically active and inactive members, with a subset involved in synthesis and degradation of other cyclic dinucleotides. Here, we summarize current knowledge of sequence and structural variations that underpin the functional diversification of cyclic di-GMP turnover proteins. Moreover, we highlight that superfamily diversification is not restricted to cyclic di-GMP signaling domains, as particular DHH/DHHA1 domain and HD domain proteins have been shown to act as cyclic di-AMP phosphodiesterases. We conclude with a consideration of the current limitations that such diversity of action places on bioinformatic prediction of the roles of GGDEF, EAL, and HD-GYP domain proteins.KEYWORDS cyclic dinucleotide second messenger, GGDEF domain, EAL domain, HD-GYP domain, DHH/DHHA1 domain, cyclic GAMP, cyclic di-AMP, cyclic di-GMP, second messenger T he dinucleotide cyclic di-GMP is the most abundant second messenger in bacteria. It promotes the environmental lifestyle switch between sessility and motility, as well as the host-related lifestyle switch between acute and chronic/benign infection. A hallmark of the cyclic di-GMP signaling network is an apparent redundancy of cyclic di-GMP turnover proteins encoded in one genome. However, many of these proteins have distinct N-terminal sensing and signaling domains, suggesting that their activities in cyclic di-GMP turnover respond posttranslationally to various (and different) intraand extracellular signals. In gross terms, the number of cyclic di-GMP turnover proteins is linearly correlated with genome size within the different bacterial phyla, with Thermotogae having one of the highest cyclic di-GMP-related "IQs," the density of enzymes per megabase pair, with some species harboring over 100 cyclic di-GMP turnover proteins (http://www.ncbi.nlm.nih.gov/Complete_Genomes/c-di-GMP.html). As in other domain superfamilies, extensive sequence diversity exists. Here, we review the knowledge on the translation of sequence diversity of cyclic di-GMP turnover proteins into functional diversity. We conclude by discussing whether and how a unified nomenclature for cyclic di-GMP turnover proteins can be established.

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“…The activity of HD-GYP from Borrelia burgdorferi was shown to be enhanced by the addition of Mn II (42). By contrast, the crystal structure of HD-GYP from Bdellovibrio bacteriovorus was solved with a diiron cluster (7), and the HD-[HD-GYP] from Vibrio cholerae was suggested to be active in the diiron form (43). No functions have been assigned to the proteins in the YqeK clade (in light green; SI Appendix, Fig.…”

Section: Resultsmentioning

confidence: 99%

Organophosphonate-degrading PhnZ reveals an emerging family of HD domain mixed-valent diiron oxygenases

Wörsdörfer¹,

Lingaraju²,

Yennawar

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

106

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The founding members of the HD-domain protein superfamily are phosphohydrolases, and newly discovered members are generally annotated as such. However, myo-inositol oxygenase (MIOX) exemplifies a second, very different function that has evolved within the common scaffold of this superfamily. A recently discovered HD protein, PhnZ, catalyzes conversion of 2-amino-1-hydroxyethylphosphonate to glycine and phosphate, culminating a bacterial pathway for the utilization of environmentally abundant 2-aminoethylphosphonate. Using Mössbauer and EPR spectroscopies, X-ray crystallography, and activity measurements, we show here that, like MIOX, PhnZ employs a mixed-valent Fe II /Fe III cofactor for the O 2 -dependent oxidative cleavage of its substrate. Phylogenetic analysis suggests that many more HD proteins may catalyze yet-unknown oxygenation reactions using this hitherto exceptional Fe II /Fe III cofactor. The results demonstrate that the catalytic repertoire of the HD superfamily extends well beyond phosphohydrolysis and suggest that the mechanism used by MIOX and PhnZ may be a common strategy for oxidative C-X bond cleavage.

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An HD-GYP Cyclic Di-Guanosine Monophosphate Phosphodiesterase with a Non-Heme Diiron–Carboxylate Active Site

Cited by 22 publications

References 7 publications

Structural Basis of Functional Diversification of the HD-GYP Domain Revealed by the Pseudomonas aeruginosa PA4781 Protein, Which Displays an Unselective Bimetallic Binding Site

Structural Basis of Functional Diversification of the HD-GYP Domain Revealed by the Pseudomonas aeruginosa PA4781 Protein, Which Displays an Unselective Bimetallic Binding Site

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

Organophosphonate-degrading PhnZ reveals an emerging family of HD domain mixed-valent diiron oxygenases

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