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)

The docking methodology was applied to three different therapeutically interesting enzymes: human dihydroorotate dehydrogenase (DHODH), Herpes simplex virus type I thymidine kinase (HSV1 TK) and human phosphodiesterase 4 (PDE4). Programs FlexX, AutoDock and DOCK where used. The three targets represent three distinct cases. For DHODH and HSV1 TK, the binding modes of substrate and inhibitors within the active site are known, while the binding orientation of cAMP within PDE4 has been solely hypothesized. Active site of DHODH is mainly hydrophobic and the binding mode of the inhibitor brequinar was used as a template for evaluating the docking strategies. The presence of cofactors revealed to be crucial for the definition of the docking site. The HSV1 TK active site is small and polar and contains crystal water molecules and ATP. Docking of thymidine and aciclovir (ACV) within the active site was analyzed by keeping or removing water molecules. It showed the crucial role of water in predicting the binding of pyrimidines and purines. The crystal structure of PDE4 contains magnesium and zinc cations as well as catalytic water molecule but no ligand. Several docking experiments of cAMP and rolipram were performed and the results showed clear-cut dependence between the ligand orientation and the presence of metals in the active site. All three cases show specific problems of the docking methodology, depending on the character of the active site.
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PMID:Methodology and problems of protein-ligand docking: case study of dihydroorotate dehydrogenase, thymidine kinase, and phosphodiesterase 4. 1250 12

Recombinant phospholipase D (PLD) from Streptomyces chromofuscus (scPLD) has been characterized using colorimetric assays, spectroscopic investigations, and site-directed mutagenesis. scPLD, which shows phosphodiesterase activity toward a wide variety of phospholipids and phosphatase activity toward p-nitrophenyl phosphate, exhibits a visible absorption band with lambda(max) at 570 nm. Metal ion analysis performed by inductively coupled plasma mass spectroscopy shows the presence of approximately 1 equivalent of iron, 0.27 equivalent of manganese, and 0.1 equivalent of zinc per mole of protein as isolated. The metal ion content coupled with the visible absorption feature is compatible with the presence of Fe(3+)-tyrosinate coordination. When scPLD was dialyzed against solutions containing Mn(2+), Zn(2+) or EDTA, the Fe(3+) content was reduced to variable extents, and the residual specific activity correlated well with the residual iron content. Sequence homology with metal ion binding motifs in known alkaline phosphatases and purple acid phosphatase from red kidney bean shows that most of the residues involved in metal ion coordination are conserved among all the sequences considered. Mutation of some of these conserved residues (C123A, D151A, Y154F, and H391A) produced enzymes lacking iron with dramatically reduced PLD activity but little change in secondary structure or ability to bind to small unilamellar vesicles of phosphatidylcholine (with Ba(2+)) or phosphatidic acid. We suggest that scPLD is a member of a family of phosphodiesterase/phosphatases with structural and mechanistic similarity to iron-dependent purple acid phosphatases.
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PMID:An iron-dependent bacterial phospholipase D reminiscent of purple acid phosphatases. 1251 26

A phosphodiesterase was purified from the venom of the snake Bothrops alternatus by a combination of gel filtration and ion exchange chromatographies. In SDS-PAGE, the enzyme gave a single band with a molecular mass of 105 kDa, which was unaltered in the presence of beta-mercaptoethanol, indicating that the protein contained no subunits. A single protein band was also observed in native PAGE. There were no contaminating 5'-nucleotidase, alkaline phosphatase and protease activities. The enzyme was recognized by commercial bothropic antiserum and gave a single band in immunoblotting. The enzyme had a pH optimum in the range of 7.5-9.5 and the optimum temperature was 60 degrees C, with activity being rapidly lost within 1 min at > or = 70 degrees C. The Km of the enzyme was 2.69 mM. PDE activity was potentiated by cobalt and, to a lesser extent, by calcium, whereas copper, manganese, zinc, EDTA, and beta-mercaptoethanol were inhibitory. These properties show that this enzyme is very similar to that isolated from other snake venoms.
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PMID:Purification and characterization of a phosphodiesterase from Bothrops alternatus snake venom. 1263 51

Alkaline sphingomyelinase (alk-SMase) hydrolyzes dietary sphingomyelin and generates sphingolipid messengers in the gut. In the present study, we purified the enzyme, identified a part of the amino acid sequence, and found a cDNA in the GenBank coding for the protein. The cDNA contains 1841 bp, and the open reading frame encodes 458 amino acids. Transient expression of the cDNA linked to a Myc tag in COS-7 cells increased alk-SMase activity in the cell extract by 689-fold and in the medium by 27-fold. High activity was also identified in the anti-Myc immunoprecipitated proteins and the proteins cross-reacted with anti-human alk-SMase. Northern blotting of human intestinal tissues found high levels of alk-SMase mRNA in the intestine and liver. The amino acid sequence shared no similarity with acid and neutral SMases but was related to the ecto-nucleotide phosphodiesterase (NPP) family with 30-36% identity to human NPPs. Alk-SMase has a predicted signal peptide domain at the N terminus and a signal anchor domain at the C terminus. The ion-binding sites and the catalytic residue of NPPs were conserved, but the substrate specificity domain was modified. Alk-SMase had no detectable nucleotidase activity, but its activity against sphingomyelin could be inhibited by orthovanadate, imidazole, and ATP. In contrast to NPPs, alk-SMase activity was not stimulated by divalent metal ions but inhibited by Zn2+. Differing from NPP2, the alk-SMase cleaved phosphocholine but not choline from lysophosphatidylcholine. Phylogenetic tree indicated that the enzyme is a new branch derived from the NPP family. Two cDNA sequences of mouse and rat that shared 83% identity to human alk-SMase were identified in the GenBank. In conclusion, we identified the amino acid and cDNA sequences of human intestinal alk-SMase, and found that it is a novel ecto-enzyme related to the NPP family with specific features essential for its SMase activity.
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PMID:Identification of human intestinal alkaline sphingomyelinase as a novel ecto-enzyme related to the nucleotide phosphodiesterase family. 1288 74

An alkaline 5'-phosphodiesterase (5'-PDE) from barley (Hordeum distichum) malt sprouts was partially purified by thermal treatment and acetone precipitation to diminish phosphomonoesterase (PME) activity. 5'-PDE was purified 40-fold to a specific activity of 30 U mg(-1) protein with a final yield of about 32%. With synthetic substrate, the enzyme had an optimum pH of 8.9, maximum activity at 70 degrees C over 10 min, and a Km of 0.26 mM. The partially purified enzyme was activated by 10 mM Mg2+ up to 168% of the original activity, while Zn2+, Mn2+ and Cu2+ ions, chelating agent (EDTA) and NaN3 (1-10 mM), and 5'-ribonucleotides (1-5 mM) were inhibitory. Final enzyme preparation was stable over 8 d at 4 degrees C), at 70 degrees C for up to 120 min and without loss of activity over 90 d at -18 degrees C.
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PMID:Partial purification and biochemical characterization of alkaline 5'-phosphodiesterase from barley malt sprouts. 1288 21

Changes in human endometrium are essential to allow the establishment of pregnancy. These changes are induced in vivo by progesterone, and include appearance within the tissue of a specific uterine natural killer cell, characterized by an abundant expression of CD56. Changes also occur in the stromal cells, which undergo a characteristic decidualization reaction. Decidualized stromal cells are derived from the fibroblast-like cells within the endometrium, which maintain their progesterone receptors in the presence of progesterone. Prolonged exposure to progesterone induces a rounded cell characterized by release of prolactin and insulin-like growth factor binding protein-1 (IGFBP-1), and expression of tissue factor. Additional changes include the secretion of interleukin (IL)-15, vascular endothelial growth factor, and surface expression of zinc dependent metalloproteinases such as CD10 and CD13. In vitro, elevated intracellular cAMP as well as progesterone is necessary for decidualization. In vivo, these conditions may be provided by progesterone from the corpus luteum, by prostaglandin E, a stimulator of adenyl cyclase, and relaxin, which has recently been shown to be a phosphodiesterase inhibitor. Given the co-distribution of uterine natural killer cells and decidualized stromal cells, a mutual interaction might provide the correct regulatory environment for successful implantation, and penetration of the maternal blood vessels by trophoblastic cells.
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PMID:Decidualization of the human endometrial stromal cell: an enigmatic transformation. 1456 82

Anandamide (N-arachidonoylethanolamine) is known to be an endogenous ligand of cannabinoid and vanilloid receptors. Its congeners (collectively referred to as N-acylethanolamines) also show a variety of biological activities. These compounds are principally formed from their corresponding N-acyl-phosphatidylethanolamines by a phosphodiesterase of the phospholipase D-type in animal tissues. We purified the enzyme from rat heart, and by the use of the sequences of its internal peptides cloned its complementary DNAs from mouse, rat, and human. The deduced amino acid sequences were composed of 393-396 residues, and showed that the enzyme has no homology with the known phospholipase D enzymes but is classified as a member of the zinc metallohydrolase family of the beta-lactamase fold. As was overexpressed in COS-7 cells, the recombinant enzyme generated anandamide and other N-acylethanolamines from their corresponding N-acyl-phosphatidylethanolamines at comparable rates. In contrast, the enzyme was inactive with phosphatidylcholine and phosphatidylethanolamine. Assays of the enzyme activity and the messenger RNA and protein levels revealed its wide distribution in murine organs with higher contents in the brain, kidney, and testis. These results confirm that a specific phospholipase D is responsible for the generation of N-acylethanolamines including anandamide, strongly suggesting the physiological importance of lipid molecules of this class.
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PMID:Molecular characterization of a phospholipase D generating anandamide and its congeners. 1463 25

Zinc (Zn), an essential trace element, and its complexes have recently been known to exhibit insulinomimetic activities. However, the action mechanism of Zn(II) has yet been obscure. The purpose of the present study was to estimate the action mechanism of the Zn(II) complexes. We found first that Zn given in the chemical forms such as Zn(maltolate)2 and Zn(threoninate)2 complexes is highly uptaken in the isolated rat adipocytes compared with that of Zn(picolinate)2. Then, the action mechanism for the insulinomimetic activities was examined in terms of free fatty acid release from the adipocytes. Four Zn(II) compounds, ZnSO4, Zn(picolinate)2, Zn(maltolate)2, and Zn(threoninate)2, inhibited the free fatty acid release from the adipocytes treated with epinephrine (adrenaline). By using several inhibitors for fatty acids and glucose metabolisms in the adipocytes, the following results were obtained. (1) Zn(picolinic acid)2 complex acts on the insulin receptor and PI3-k, which relate to the glucose uptake, as indicated by the experiments using hydroxy-2-naphthalenylmethyl phosphonic acid tris acetoxy methyl ester (HNMPA-(AM)3) and wortmannin, respectively. (2) ZnSO4, and Zn(maltolate)2 and Zn(threoninate)2 complexes affect a glucose transporter 4 (GLUT 4), which is involved in the glucose uptake as indicated by the results using cytochalasin B. (3) Four Zn(II) compounds affect the activation of the phosphodiesterase as indicated by the experiments using cilostamide. These results indicate that the Zn(II) compounds promote the glucose uptake into the adipocytes by affecting at least three sites in the adipocytes, which in turn normalize the blood glucose levels in the experimental diabetic animals.
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PMID:The action mechanism of zinc(II) complexes with insulinomimetic activity in rat adipocytes. 1517 82

Zinc phosphodiesterase (ZiPD) is a member of the metallo-beta-lactamase family with a binuclear zinc binding site. As an experimental attempt to identify the metal ligands of Escherichia coli ZiPD and to investigate their function in catalysis, we mutationally exchanged candidate metal coordinating residues and performed kinetic and X-ray absorption spectroscopic analysis of the mutant proteins. All mutants (H66E, H69A, H141A, D212A, D212C, H231A, H248A, and H270A) show significantly lower catalytic rates toward the substrate bis(p-nitrophenyl)phosphate. Substrate binding, represented by the kinetic value K', remains unchanged for six mutants, whereas it is increased 3-4-fold for H231A and H270A. Accordingly, these two residues are supposed to be involved in substrate binding, whereas the others are more important for catalytic turnover and thus are assumed to be involved in zinc ligation. Structural insight into the metal binding of D212 was gained by zinc K-edge extended X-ray absorption fine structure (EXAFS). The sulfur coordination number of the cysteine mutant was found to be 1, demonstrating binding to both zinc metals in a bridging mode. Taken together with two residues from a strictly conserved sequence region within the metallo-beta-lactamase family, the metal site of ZiPD is proposed with H64, H66, and H141 coordinating ZnA, D68, H69, and H248 coordinating ZnB, and D212 bridging both metals. Surprisingly, the same coordination sphere is found in glyoxalase II. This is further substantiated by comparable EXAFS spectra of both native enzymes. This is the first example of the same metal site in two members of the metallo-beta-lactamase domain proteins catalyzing different reactions. The kinetic analysis of mutants provides unexpected insights into the reaction mechanism of ZiPD.
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PMID:Identification of metal binding residues for the binuclear zinc phosphodiesterase reveals identical coordination as glyoxalase II. 1530 36

The elaC gene of Escherichia coli encodes a binuclear zinc phosphodiesterase (ZiPD). ZiPD homologs from various species act as3' tRNA processing endoribonucleases, and although the homologous gene in Bacillus subtilis is essential for viability [EMBO J. 22(2003) 4534], the physiological function of E. coli ZiPD has remained enigmatic. In order to investigate the function of E. coli ZiPDwe generated and characterized an E. coli elaC deletion mutant. Surprisingly, the E. coli elaC deletion mutant was viable and had wild-type like growth properties. Microarray-based transcriptional analysis indicated expression of the E. coli elaC gene at basal levels during aerobic growth. The elaC gene deletion had no effect on the expression of genes coding for RNases or amino-acyl tRNA synthetases or any other gene among a total of > 1300 genes probed. 2D-PAGE analysis showed that the elaC mutation, like-wise, had no effect on the proteome. These results strengthen doubts about the involvement of E. coli ZiPD in tRNA maturation and suggest functional diversity within the ZiPD/ElaC1 protein family. In addition to these unexpected features of the E. coli elaC deletion mutant, a sequence comparison of ZiPD (ElaC1) proteins revealed specific regions for either enterobacterial or mammalian ZiPD (ElaC1) proteins.
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PMID:Characterization of an Escherichia coli elaC deletion mutant. 1530 84


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