Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bacteria use extracellular levels of small diffusible autoinducers to estimate local cell-density (quorum-sensing) and to regulate complex physiological processes. The quorum-sensing signal transduction pathway of Xanthomonas spp. phytopathogens has special features that distinguish it from that of other pathogens. This pathway consists of RpfF, necessary for the production of the unique autoinducer 'diffusible signalling factor' (DSF), and RpfC and RpfG, a two-component system necessary for the DSF-dependent production of extracellular pathogenicity factors and cellular dispersion. Yeast two-hybrid and direct in vitro assays were used to identify interactions involving the Rpf group of proteins. We show that RpfC, a protein consisting of N-terminal transmembrane, histidine kinase, response-regulator and C-terminal histidine phosphotransfer domains interacts with both RpfG, a protein consisting of an N-terminal response regulator domain and a C-terminal HD-GYP domain, and with RpfF. We also show that RpfC interacts with the only known homologue of 'conditioned medium factor', which is involved in quorum-sensing in Dictyostelium discoideum under conditions of nutritional stress. Furthermore, RpfCG is shown to interact with a second two-component system made up of NtrB and NtrC homologues. Finally we show that the recently characterized HD-GYP phosphodiesterase domain of RpfG interacts directly with diguanylate cyclase GGDEF domain-containing proteins coded by the Xanthomonas axonopodis pv. citri genome, which in other bacteria produce cyclic diGMP, an important second messenger involved in the regulation of complex bacterial processes including biofilm production, virulence and motility. These results demonstrate a direct physical linkage between quorum-sensing and cyclic diGMP signalling pathways in bacteria.
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PMID:The HD-GYP domain of RpfG mediates a direct linkage between the Rpf quorum-sensing pathway and a subset of diguanylate cyclase proteins in the phytopathogen Xanthomonas axonopodis pv citri. 1702 May 86

Cyclic nucleotide monophosphate (cNMP) hydrolysis in bacteria and eukaryotes is brought about by distinct cNMP phosphodiesterases (PDEs). Since these enzymes differ in amino acid sequence and properties, they have evolved by convergent evolution. Cyclic NMP PDEs cleave cNMPs to NMPs, and the Rv0805 gene product is, to date, the only identifiable cNMP PDE in the genome of Mycobacterium tuberculosis. We have shown that Rv0805 is a cAMP/cGMP dual specificity PDE, and is unrelated in amino acid sequence to the mammalian cNMP PDEs. Rv0805 is a dimeric, Fe(3+)-Mn(2+) binuclear PDE, and mutational analysis demonstrated that the active site metals are co-ordinated by conserved aspartate, histidine and asparagine residues. We report here the structure of the catalytic core of Rv0805, which is distantly related to the calcineurin-like phosphatases. The crystal structure of the Rv0805 dimer shows that the active site metals contribute to dimerization and thus play an additional structural role apart from their involvement in catalysis. We also present the crystal structures of the Asn97Ala mutant protein that lacks one of the Mn(2+) co-ordinating residues as well as the Asp66Ala mutant that has a compromised cAMP hydrolytic activity, providing a structural basis for the catalytic properties of these mutant proteins. A molecule of phosphate is bound in a bidentate manner at the active site of the Rv0805 wild-type protein, and cacodylate occupies a similar position in the crystal structure of the Asp66Ala mutant protein. A unique substrate binding pocket in Rv0805 was identified by computational docking studies, and the role of the His140 residue in interacting with cAMP was validated through mutational analysis. This report on the first structure of a bacterial cNMP PDE thus significantly extends our molecular understanding of cAMP hydrolysis in class III PDEs.
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PMID:Structural and biochemical analysis of the Rv0805 cyclic nucleotide phosphodiesterase from Mycobacterium tuberculosis. 1705 28

The Escherichia coli AcpH acyl carrier protein phosphodiesterase (also called ACP hydrolyase) is the only enzyme known to cleave a phosphodiester-linked post-translational protein modification. AcpH hydrolyzes the link between 4'-phosphopanthetheine and the serine-36 side chain of acyl carrier protein (ACP). Although the existence of this enzyme activity has long been known, study of the enzyme was hampered by its recalcitrant properties and scarcity. We recently isolated the gene encoding AcpH and have produced the recombinant enzyme in quantity (Thomas, J., and Cronan, J. E., (2005) J. Biol. Chem. 280, 34675-34683), thus allowing the first studies of its reaction mechanism. AcpH requires Mn2+ for activity, and thus, we focused on the metal binding ligands in order to locate the active site. Bioinformatic investigations indicated that AcpH and its homologues were weakly related to a phosphodiesterase of known structure, the hydrolyase domain of the bifunctional bacterial protein, SpoT, suggesting that AcpH is a member of the HD family of phosphatases/ phosphodiesterases despite lacking the characteristic histidine of the motif. Indeed, we found that AcpH could be convincingly modeled on the SpoT structure with acceptable parameters, which allowed the identification of putative metal binding ligands. These were then tested by site-directed mutagenesis. Mutagenic removal of any of the putative ligands resulted in a severe or total loss of phosphodiesterase activity. In two cases, the H6Q and D24N proteins, the residual activities could be markedly stimulated by addition of high Mn2+ concentrations, thereby demonstrating a role for these residues in metal binding. We conclude that AcpH is a member of the HD protein family despite the lack of the signature histidine residue.
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PMID:Acyl carrier protein phosphodiesterase (AcpH) of Escherichia coli is a non-canonical member of the HD phosphatase/phosphodiesterase family. 1719 82

Oxidative stress is a principal cause of DNA damage, and mechanisms to repair this damage are among the most highly conserved of biological processes. Oxidative stress is also used by phagocytes to attack bacterial pathogens in defence of the host. We have identified and characterised two apurinic/apyrimidinic (AP) endonuclease paralogues in the human pathogen Neisseria meningitidis. The presence of multiple versions of DNA repair enzymes in a single organism is usually thought to reflect redundancy in activities that are essential for cellular viability. We demonstrate here that these two AP endonuclease paralogues have distinct activities in DNA repair: one is a typical Neisserial AP endonuclease (NApe), whereas the other is a specialised 3'-phosphodiesterase Neisserial exonuclease (NExo). The lack of AP endonuclease activity of NExo is shown to be attributable to the presence of a histidine side chain, blocking the abasic ribose-binding site. Both enzymes are necessary for survival of N. meningitidis under oxidative stress and during bloodstream infection. The novel functional pairing of NExo and NApe is widespread among bacteria and appears to have evolved independently on several occasions.
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PMID:AP endonuclease paralogues with distinct activities in DNA repair and bacterial pathogenesis. 1731 83

The human orthologue of the Drosophila prune protein (h-Prune) is an interaction partner and regulator of the metastasis suppressor protein NM23-H1 (non-metastatic protein 23). Studies on a cellular breast-cancer model showed that inhibition of the cAMP-specific PDE (phosphodiesterase) activity of h-Prune lowered the incidence of metastasis formation, suggesting that inhibition of h-Prune could be a therapeutic approach towards metastatic tumours. H-Prune shows no sequence similarity with known mammalian PDEs, but instead appears to belong to the DHH (Asp-His-His) superfamily of phosphoesterases. In order to investigate the structure and molecular function of h-Prune, we expressed recombinant h-Prune in a bacterial system. Through sequence analysis and limited proteolysis, we identified domain boundaries and a potential coiled-coil region in a C-terminal cortexillin homology domain. We found that this C-terminal domain mediated h-Prune homodimerization, as well as its interaction with NM23-H1. The PDE catalytic domain of h-Prune was mapped to the N-terminus and shown to be active, even when present in a monomeric form. Our findings indicate that h-Prune is composed of two independent active sites and two interaction sites for the assembly of oligomeric signalling complexes.
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PMID:Domain mapping on the human metastasis regulator protein h-Prune reveals a C-terminal dimerization domain. 1765 25

The molecular bases for phosphodiesterase 5 (PDE5) catalytic-site affinity for cyclic guanosine monophosphate (cGMP) and potency of inhibitors are poorly understood. Cocrystal structures of PDE5 catalytic (C) domain with inhibitors reveal a hydrogen bond and hydrophobic interactions with Tyr-612, hydrogen bonds with Gln-817, a hydrophobic clamp formed by Phe-820 and Val-782, and contacts with His-613, Leu-765, and Phe-786 [Sung et al. (2003) Nature 425, 98-102; Huai et al. (2004) J. Biol. Chem. 279, 13095-13101]. Present results of point mutations of full-length PDE5 showed that maximum catalysis was decreased 2650-fold in H613A and 55-fold in F820A. Catalytic-site affinities for cGMP, vardenafil, sildenafil, tadalafil, or 3-isobutyl-1-methylxanthine (IBMX) were respectively weakened 14-, 123-, 30-, 51-, and 43-fold for Y612A; 63-, 511-, 43-, 95- and 61-fold for Q817A; and 59-, 448-, 71-, 137-, and 93-fold for F820A. The data indicate that these three amino acids are major determinants of affinity for cGMP and potency of selective and nonselective inhibitors, and that higher vardenafil potency over sildenafil and tadalafil results from stronger contacts with Tyr-612, Gln-817, and Phe-820. Affinity of V782A for cGMP, vardenafil, sildenafil, tadalafil, or IBMX was reduced 5.5-, 23-, 10-, 3-, and 12-fold, respectively. Change in affinity for cGMP, vardenafil, sildenafil, or IBMX in Y612F, H613A, L765A, or F786A was less, but affinity of H613A or F786A for tadalafil was weakened 37- and 17-fold, respectively. The results quantify the role of PDE5 catalytic-site residues for cGMP and inhibitors, indicate that Tyr-612, Gln-817, and Phe-820 are the most important cGMP or inhibitor contacts studied, and identify residues that contribute to selectivity among different classes of inhibitors.
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PMID:Critical amino acids in phosphodiesterase-5 catalytic site that provide for high-affinity interaction with cyclic guanosine monophosphate and inhibitors. 1797 1

In Escherichia coli, exonuclease I (ExoI) is a monomeric processive 3'-5' exonuclease that degrades single-stranded DNA. The enzyme has been implicated as primarily being involved in repairing frameshift mutations. The structure of the enzyme has previously been determined in a hexagonal space group at 2.4 A resolution. Here, the structure of ExoI in complex with a nucleotide product, thymidine 5'-monophosphate, is described in an orthorhombic space group at 1.5 A resolution. This new high-resolution structure provides some insight into the interactions involved in binding a nucleotide product. The conserved active site contains a unique metal-binding position when compared with orthologous sites in the Klenow fragment, T4 DNA polymerase and dnaQ. This unique difference is proposed to be a consequence of the repositioning of an important histidine, His181, away from the active site.
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PMID:Structure of Escherichia coli exonuclease I in complex with thymidine 5'-monophosphate. 1821 21

Phosphodiesterase plays an important role in regulating inflammatory pathways and T cell function. The development of phosphodiesterase 7 inhibitor may give better efficacy profile over phosphodiesterase 4 inhibitors. However, the recombinant phosphodiesterase 7 is required in large quantity for high-throughput screening of new drugs by in vitro enzymatic assays. In the present study, recombinant human PDE7A1 was expressed in Dictyostelium discoideum under the control of constitutively active actin-15 promoter. The cytosolic localization of the expressed protein was confirmed by immunofluorescence studies. Upto 2 mg of recombinant protein was purified using His-Tag affinity column chromatography followed by ion-exchange Resource Q column purification. The recombinant protein expressed in D. discoideum followed Michaelis-Menten kinetics similar to the protein expressed in mammalian system and showed no major changes in affinity to substrate or inhibitors. Thus, our study clearly demonstrates a robust expression system for successful bulk production of pharmacologically active isoform of human PDE7A1 required for high-throughput assays.
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PMID:Purification of recombinant human phosphodiesterase 7A expressed in Dictyostelium discoideum. 1854 17

Binuclear metallophosphoesterases are an enzyme superfamily defined by a shared fold and a conserved active site. Although many family members have been characterized biochemically or structurally, the physiological substrates are rarely known, and the features that determine monoesterase versus diesterase activity are obscure. In the case of the dual phosphomonoesterase/diesterase enzyme CthPnkp, a phosphate-binding histidine was implicated as a determinant of 2',3'-cyclic nucleotide phosphodiesterase activity. Here we tested this model by comparing the catalytic repertoires of Mycobacterium tuberculosis Rv0805, which has this histidine in its active site (His(98)), and Escherichia coli YfcE, which has a cysteine at the equivalent position (Cys(74)). We find that Rv0805 has a previously unappreciated 2',3'-cyclic nucleotide phosphodiesterase function. Indeed, Rv0805 was 150-fold more active in hydrolyzing 2',3'-cAMP than 3',5'-cAMP. Changing His(98) to alanine or asparagine suppressed the 2',3'-cAMP phosphodiesterase activity of Rv0805 without adversely affecting hydrolysis of bis-p-nitrophenyl phosphate. Further evidence for a defining role of the histidine derives from our ability to convert the inactive YfcE protein to a vigorous and specific 2',3'-cNMP phosphodiesterase by introducing histidine in lieu of Cys(74). YfcE-C74H cleaved the P-O2' bond of 2',3'-cAMP to yield 3'-AMP as the sole product. Rv0805, on the other hand, hydrolyzed either P-O2' or P-O3' to yield a mixture of 3'-AMP and 2'-AMP products, with a bias toward 3'-AMP. These reaction outcomes contrast with that of CthPnkp, which cleaves the P-O3' bond of 2',3'-cAMP to generate 2'-AMP exclusively. It appears that enzymic features other than the phosphate-binding histidine can influence the orientation of the cyclic nucleotide and thereby dictate the choice of the leaving group.
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PMID:A phosphate-binding histidine of binuclear metallophosphodiesterase enzymes is a determinant of 2',3'-cyclic nucleotide phosphodiesterase activity. 1875 71

Carbon-phosphorus lyase is a multienzyme system encoded by the phn operon that enables bacteria to metabolize organophosphonates when the preferred nutrient, inorganic phosphate, is scarce. One of the enzymes encoded by this operon, PhnP, is predicted by sequence homology to be a metal-dependent hydrolase of the beta-lactamase superfamily. Screening with a wide array of hydrolytically sensitive substrates indicated that PhnP is an enzyme with phosphodiesterase activity, having the greatest specificity toward bis(p-nitrophenyl)phosphate and 2',3'-cyclic nucleotides. No activity was observed toward RNA. The metal ion dependence of PhnP with bis(p-nitrophenyl)phosphate as substrate revealed a distinct preference for Mn(2+) and Ni(2+) for catalysis, whereas Zn(2+) afforded poor activity. The three-dimensional structure of PhnP was solved by x-ray crystallography to 1.4 resolution. The overall fold of PhnP is very similar to that of the tRNase Z endonucleases but lacks the long exosite module used by these enzymes to bind their tRNA substrates. The active site of PhnP contains what are probably two Mn(2+) ions surrounded by an array of active site residues that are identical to those observed in the tRNase Z enzymes. A second, remote Zn(2+) binding site is also observed, composed of a set of cysteine and histidine residues that are strictly conserved in the PhnP family. This second metal ion site appears to stabilize a structural motif.
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PMID:Structure of PhnP, a phosphodiesterase of the carbon-phosphorus lyase pathway for phosphonate degradation. 1936 88


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