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At least 649 records · Page 36Linked to original sources
PubMed1978

Formation of 1-beta-D-arabinofuranosylcytosine diphosphate choline in cultured human leukemic RPMI 6410 cells.

When incubated with 1-beta-D-arabinofuranosylcytosine (ara-C), RPMI 6410 cells formed a hitherto unrecognized ara-C metabolite, 1-beta-D-arabinofuranosylcytosine diphosphate choline. This compound was characterized by (a) chromatographic behavior, (b) chemical and enzymatic hydrolysis, (c) phosphorus content, and (d) incorporation of [5-3H]ara-C and [methyl-14C]choline. Formation of 1-beta-D-arabinofuranosylcytosine diphosphate choline by RPMI 6410 cells was enhanced in the presence of 3-deazauridine (DU) and was preceded by that of 1-beta-D-arabinofuranosylcytosine triphosphate. The antiproliferative effects of ara-C and DU toward RPMI 6410 cells were potentiated when the agents were present together. The anabolism of ara-C during a 24-hr interval of culture was markedly enhanced by the presence of DU; cellular concentrations of 1-beta-D-arabinofuranosylcytosine triphosphate and 1-beta-D-arabinofuranosylcytosine diphosphate choline were 5- and 15-fold higher than those in the absence of DU. This enhancement appears to be the basis of the potentiation of cytotoxicity resulting from combination of the agents. Pretreatment of RPMI 6410 cells with DU resulted in enhanced rates of cellular uptake of ara-C. ara-C uptake under these circumstances was blocked by the inhibitor of nucleoside transport, nitrobenzylthioinosine.

3-Deazauridine↗
PubMed1978

Formation of 1-beta-D-arabinofuranosylcytosine diphosphate choline in neoplastic and normal cells.

1-beta-D-Arabinofuranosylcytosine diphosphate choline was formed from 1-beta-D-arabinofuranosylcytosine (ara-C) during incubation in vitro of peripheral myeloblasts from patients with acute myelogenous leukemia and cultured cells (nonleukemic human lymphocytes, mouse lymphoma L5178Y, and HeLa); as well, 1-beta-D-arabinofuranosylcytosine diphosphate choline was formed in vivo in mouse leukemia L1210 cells and mouse liver. 3-Deazauridine enhanced the anabolism of ara-C in nonleukemic lymphocytes in vitro and leukemia L1210 cells in vivo but did not influence ara-C anabolism in the other cell types. In acute myelogenous leukemia myeloblasts incubated in vitro with ara-C, concentrations of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate were maximal after 8 hr of incubation and formation of the latter preceded that of 1-beta-D-arabinofuranosylcytosine diphosphate choline.

3-Deazauridine↗
PubMed1986

A guanine nucleotide-dependent phosphatidylinositol 4,5-diphosphate phospholipase C in cells transformed by the v-fms and v-fes oncogenes.

The metabolism of phosphatidylinositol (PtdIns) was studied in a mink lung epithelial cell line and its subclones transformed by feline sarcoma viruses containing either the v-fms or v-fes oncogenes. The transformed cell lines had a higher rate of PtdIns turnover but did not have elevated levels of phosphorylated PtdIns species or PtdIns kinase activity. Significantly higher specific activities of a guanine nucleotide-activated PtdIns-4,5-diphosphate phospholipase C were detected in both transformed cell lines (F3CL7(v-fes), 55 pmol/min/mg of protein and G2M(v-fms), 18 pmol/min/mg of protein) as compared to the nontransformed parental cell line (CCL64, 2 pmol/min/mg of protein). The guanine nucleotide-stimulated phospholipase C activity was specific for PtdIns-4,5-diphosphate, and the water-soluble hydrolysis product was inositol 1,4,5-triphosphate. Both GTP and nonhydrolyzable GTP analogs activated the phospholipase C, whereas ATP was weakly effective and GDP was inactive. The phospholipase C activity was maximally active in the presence of 9 mM sodium cholate, had a sharp pH optimum of pH 6.5, and was not activated by calcium although hydrolysis was inhibited by high concentrations of EDTA. These data point to enhanced production of diacylglycerol and inositol 1,4,5-triphosphate second messengers in transformed cells due to the activation of guanine nucleotide-dependent PtdIns-4,5-diphosphate-specific phospholipase C and suggest that the generation of aberrant hormonally independent signals is associated with cell transformation by oncogenes encoding tyrosine-specific protein kinases.

1-Phosphatidylinositol 4-Kinase↗
PubMed1986

Inactivation of NAD-dependent isocitrate dehydrogenase by affinity labeling of the allosteric ADP site by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate.

A new reactive ADP analogue has been synthesized: 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-diphosphate (2-BDB-TADP). Reaction of ADP with m-chloroperoxybenzoic acid gave ADP 1-oxide, which was treated with NaOH, followed by reaction with carbon disulfide to yield 2-thioadenosine 5'-diphosphate. The final product was synthesized by condensation of 2-thioadenosine 5'-diphosphate with 1,4-dibromobutanedione. Reaction of pig heart NAD-specific isocitrate dehydrogenase with this nucleotide analogue (0.4 mM) causes a time-dependent loss of activity to a limiting value of 75% inactivation. The rate constant for inactivation exhibits a nonlinear dependence on the concentration of 2-BDB-TADP, with kmax = 0.021 min-1 and KI = 0.067 mM. Complete protection against inactivation by 0.2 mM 2-BDB-TADP is provided by ADP + Mn2+, but not by Mn2+ alone, isocitrate, alpha-ketoglutarate, or NAD. Incorporation of 2-BDB-TADP is proportional to the extent of inactivation, reaching 1 mol of reagent/mol of enzyme subunit when the enzyme is maximally inactivated. However, when inactivation is totally prevented by incubation with 2-BDB-TADP in the presence of ADP and Mn2+, 0.5 mol of reagent/mol of subunit is still incorporated, suggesting that inactivation may be attributed to 0.5 mol of reagent/mol of average subunit. In the native enzyme, the Km for total isocitrate is 1.8 mM and is decreased 6-fold to 0.3 mM in the presence of 1 mM ADP, whereas in the modified enzyme, with 25% residual activity, the Km for total isocitrate is about the same in the absence (2.0 mM) or presence (1.8 mM) of ADP. These results indicate that 2-BDB-TADP acts as an affinity label of the ADP allosteric site of NAD-dependent isocitrate dehydrogenase.

Adenosine Diphosphate↗
PubMed1985

Platelet aggregation in whole blood: the role of thromboxane A2 and adenosine diphosphate.

The platelet aggregation that occurred in whole blood in response to several aggregating agents (collagen, arachidonic acid, adenosine diphosphate, adrenaline and thrombin) was measured using an Ultra-Flo 100 Whole Blood Platelet Counter. The amounts of thromboxane B2 produced were measured by radioimmunoassay. The effects of various inhibitors of thromboxane synthesis and the effects of apyrase, an enzyme that destroys adenosine diphosphate, were determined. Platelet aggregation was always accompanied by the production of thromboxane B2, and the amounts produced depended on the nature and concentration of the aggregating agent used. The various inhibitors of thromboxane synthesis--aspirin and flurbiprofen (cyclo-oxygenase inhibitors), BW755C (a cyclo-oxygenase and lipoxygenase inhibitor) and dazoxiben (a selective thromboxane synthase inhibitor)--did not markedly inhibit aggregation. Results obtained using apyrase showed that adenosine diphosphate contributed to the aggregation process, and that its role must be acknowledged when devising means of inhibiting platelet aggregation in vivo.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1980

The purification and characterization of an adenosylcobalamin-dependent ribonucleoside diphosphate reductase from Corynebacterium nephridii.

A deoxyadenosylcobalamin-dependent ribonucleoside diphosphate reductase has been purified to homogeneity from cell free extracts of Corynebacterium nephridii. Ion exchange chromatography of th extract on DEAE-Sephadex and DEAE-Bio-Gel A, followed by affinity chromatography on dGTP-Sepharose, yielded two forms of the reductase. The first reductase, which was weakly bound to the affinity column, was eluted with 0.1 M citrate buffer, pH 6.5, while the second, more tightly bound form required 2 M urea for its elution. The weakly bound form is homogeneous as judged by gel filtration, equilibrium sedimentation, and polyacrylamide gel electrophoresis. This enzyme is a dimeric protein with a molecular weight of 196,000, composed of two identical or very similar subunits with molecular weights of 100,000. The second protein appears to be a polymeric form of the reductase. Both forms of the enzyme catalyze the reduction of the four common ribonucleoside diphosphates as well as the hydrogen exchange between adenosylcobalamin and the solvent. The ribonucleoside diphosphate reductase system of C. nephridii appears to be intermediate between the more "primitive" adenosylcobalamin-dependent reductase system of Lactobacillus leichmannii and the more "advanced" nonheme iron system of Escherichia coli.

Amino Acid Sequence↗
PubMed1981

Possible regulation of ribonucleoside diphosphate reductase.

Two inhibitors of ribonucleoside diphosphate reductase (RR) (EC 1.17.4.1) in vitro were isolated from normal rat liver: they were a nondialyzable, heat-labile, high-molecular-weight ribonucleoside diphosphate reductase inhibitor (HRRI, and a dialyzable, heat-stable, low-molecular-weight ribonucleoside diphosphate reductase inhibitor (LRRI). The activities of both inhibitors varied inversely with the cell growth rate. HRRI from the cytosol fraction of rat liver was partially purified by ammonium sulfate fractionation (0 - 50%), and gel filtration on a Sepharose 6B column. It was eluted in the void volume from this column, together with ATP-hydrolyzing activity. The HRRI fraction also contained CDP kinase and CDPase activities, suggesting that HRRI is a complex of several enzymes that reduce the concentrations of the substrate of RR, CDP, and of the allosteric activator, ATP. LRRI was extracted from the cytosol of rat liver with ethanol (80% final concentration) and purified further by washing with organic solvent, and be chromatographies of Amberlite IR-45 and Dowex 50. Finally, it was identified as glucose, which was phosphorylated to glucose 6-phosphate by hexokinase present tin the RR enzyme solution ( 0 - 35% ammonium sulfate fraction of AH-130 cytosol), thus causing ATP depletion. Thus, neither inhibitor reacted directly with the RR enzyme, but both may regulate the enzyme activity in vivo by reducing the intracellular levels of substrates or cofactors.

Animals↗
PubMed1995

Mechanism of phosphate transfer by nucleoside diphosphate kinase: X-ray structures of the phosphohistidine intermediate of the enzymes from Drosophila and Dictyostelium.

Nucleoside diphosphate kinase (NDP kinase) has a ping-pong mechanism with a phosphohistidine intermediate. Crystals of the enzymes from Dictyostelium discoideum and from Drosophila melanogaster were treated with phosphoramidate, and their X-ray structures were determined at 2.1 and 2.2 A resolution, respectively. The atomic models, refined to R factors below 20%, show no conformation change relative to the free proteins. In both enzymes, the active site histidine was phosphorylated on N delta, and it was the only site of phosphorylation. The phosphate group interacts with the hydroxyl group of Tyr56 and with protein-bound water molecules. Its environment is compared with that of phosphohistidines in succinyl-CoA synthetase and in phosphocarrier proteins. The X-ray structures of phosphorylated NDP kinase and of previously determined complexes with nucleoside diphosphates provide a basis for modeling the Michaelis complex with a nucleoside triphosphate, that of the phosphorylated protein with a nucleoside diphosphate, and the transition state of the phosphate transfer reaction in which the gamma-phosphate is pentacoordinated.

Animals↗
PubMed2001

Binding of nucleotides to nucleoside diphosphate kinase: a calorimetric study.

The source of affinity for substrates of human nucleoside diphosphate (NDP) kinases is particularly important in that its knowledge could be used to design more effective antiviral nucleoside drugs (e.g., AZT). We carried out a microcalorimetric study of the binding of enzymes from two organisms to various nucleotides. Isothermal titration calorimetry has been used to characterize the binding in terms of Delta G degrees, Delta H degrees and Delta S degrees. Thermodynamic parameters of the interaction of ADP with the hexameric NDP kinase from Dictyostelium discoideum and with the tetrameric enzyme from Myxococcus xanthus, at 20 degrees C, were similar and, in both cases, binding was enthalpy-driven. The interactions of ADP, 2'deoxyADP, GDP, and IDP with the eukaryotic enzyme differed in enthalpic and entropic terms, whereas the Delta G degrees values obtained were similar due to enthalpy--entropy compensation. The binding of the enzyme to nonphysiological nucleotides, such as AMP--PNP, 3'deoxyADP, and 3'-deoxy-3'-amino-ADP, appears to differ in several respects. Crystallography of the protein bound to 3'-deoxy-3'-amino-ADP showed that the drug was in a distorted position, and was unable to interact correctly with active site side chains. The interaction of pyrimidine nucleoside diphosphates with the hexameric enzyme is characterized by a lower affinity than that with purine nucleotides. Titration showed the stoichiometry of the interaction to be abnormal, with 9--12 binding sites/hexamer. The presence of supplementary binding sites might have physiological implications.

Adenosine Diphosphate↗
PubMed1989

Deficit of uridine diphosphate galactose in galactosaemia.

The levels of uridine diphosphate galactose (UDPGal) and uridine diphosphate glucose (UDPGlc) have been determined in liver autopsy samples, erythrocytes and cultured skin fibroblasts from galactosaemic patients and compared to non-galactosaemic controls. In patients with undetectable erythrocyte galactose-1-phosphate uridyltransferase (transferase) activity, the levels of UDPGal were substantially lower than in controls. In patients with detectable transferase activity, even though in less than 1% of normal values, both UDPGal and UDPGlc levels were in the normal range. Incubation of erythrocytes from both galactosaemic patients and normal individuals with 10 mmol/L uridine increased UDPGal and UDPGlc levels several-fold, both in the presence or absence of galactose in the incubation medium. We hypothesize that a deficit of UDPGal is responsible for the late onset clinical manifestations in galactosaemia which include ovarian failure, speech defect and neurological abnormalities. We suggest that uridine administration may be of therapeutic value in raising the intracellular concentrations of UDPGal. We conclude that the transferase reaction, however small in activity, is essential for optimal UDPGal formation.

Cells, Cultured↗
PubMed1985

Uridine 5'-diphosphate glucose analogues. Inhibitors of protein glycosylation that show antiviral activity.

A series of analogues of uridine 5'-diphosphate glucose and uridine 5'-diphosphate glucosamine have been synthesized by reaction of 2,3,4,6-tetra-O-benzyl-, 2,3,4,6-tetra-O-benzoyl-, 2,3,4,6-tetra-O-acetyl-, and 2,3,4,6-tetra-O-palmitoyl-alpha-D-glucopyranose and 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranose with chlorosulfonyl isocyanate and 2',3'-O-isopropylideneuridine. Isopropylidene and acetyl groups of the resulting 5'-O-[[[[(alpha-D-glucopyranosyl)oxy]carbonyl]amino]sulfonyl] -2',3'-O-isopropylideneuridine derivatives were removed by reaction with a TFA/water (5:1) mixture and methanolic ammonia, respectively. The 5'-O-[[[[(2",3",4",6"-tetra-O-benzyl-and 2",3",4",6"-tetra-O-benzoyl-alpha-D-glucopyranosyl)oxy]carbonyl] amino]sulfonyl]-2',3'-O-isopropylideneuridine (13 and 19) and the corresponding deisopropylidenated derivatives showed antiviral activity as determined by the inhibition of the cytopathic effect induced by HSV-1 replication and by the plaque assay method. Compound 13 inhibited glycosylation of proteins in HSV-1 infected HeLa cells.

Antiviral Agents↗
PubMed1992

Membrane-bound nucleoside diphosphate kinase activity in atrial cells of frog, guinea pig, and human.

Muscarinic K+ channels in inside-out patches of atrial cells from guinea pig or rabbit can be activated by Mg(2+)-ATP in the absence of acetylcholine and GTP or GDP. The ATP-dependent activation involves a phosphorylation and is postulated to be due to the association of a membrane-bound nucleoside diphosphate kinase (NDPK) with the G protein GK: direct phosphorylation of the GK-bound GDP into GTP, catalyzed by NDPK, would result in activation of the G protein and, hence, activation of the channels. The aim of this study was to identify the presence of NDPK activity in atrial membranes by investigating the phosphate transfer between tritium-labeled nucleotides. We show that frog, guinea pig, and human atrial membranes contain a substantial NDPK activity since they catalyze the conversion from [3H]GDP+nucleoside triphosphate (NTP or NTP gamma S) to [3H]GTP (or [3H]GTP gamma S), from [3H]ADP+NTP to [3H]ATP, and from [3H]GTP+nucleoside diphosphate (NDP) to [3H]GDP. The phosphate transfer rates for the [3H]GDP+ATP to [3H]GTP conversion are 1.8, 0.5, and 2.4 mumol inorganic phosphate formation/mg per 10 minutes at 37 degrees C in frog, guinea pig, and human, respectively. The order of substrate efficiency for different NTPs was ATP greater than ITP approximately equal to GTP greater than UTP greater than CTP, which parallels the efficiency of these nucleotides in their activation of the muscarinic K+ channels. Addition of other nucleotides blocked the transphosphorylation reaction, indicating that the NTP-NDP conversion mechanism is aspecific, as is expected for an NDPK-catalyzed reaction. In conclusion, the data support the concept of NDPK involvement in the atrial muscarinic signal transduction cascade.

Adenosine Diphosphate↗
PubMed2004

Stereochemical studies on the making and unmaking of isopentenyl diphosphate in different biological systems.

To investigate the unknown stereochemical course of the reaction catalyzed by the type-II isomerase, which interconverts isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), a sample of [1,2-(13)C2]-IPP stereospecifically labelled with 2H at C2 was prepared by incubating a D2O solution of (E)-4-hydroxy-3-methyl[1,2-(13)C2]but-2-enyl diphosphate with a recombinant IspH protein of Escherichia coli in the presence of NADH as a reducing agent and flavodoxin as well as flavodoxin reductase as auxiliary proteins. As monitored by 13C-NMR spectroscopy, treatment of the deuterated IPP with either type-I or type-II IPP isomerase resulted in the formation of DMAPP molecules retaining all the 2H label of the starting material. From the known stereochemical course of the type-I isomerase-catalyzed reaction, one has to conclude that the label introduced from D2O in the course of the IspH reaction resides specifically in the H(Si)-C2 position of IPP and that the two isomerases mobilize specifically the same H(Re)-C2 ligand of their common IPP substrate. The outcome of an additional experiment, in which unlabelled IPP was incubated in D2O with the type-II enzyme, demonstrates that the two isomerases also share the same preference in selecting for their reaction the (E)-methyl group of DMAPP.

Bacillus subtilis↗
PubMed2007

Gas-phase chemistry of diphosphate anions as a tool to investigate the intrinsic requirements of phosphate ester enzymatic reactions: the [M1M2HP2O7]- ions.

Experimental studies on gaseous inorganic phosphate ions are practically nonexistent, yet they can prove helpful for a better understanding of the mechanisms of phosphate ester enzymatic processes. The present contribution extends our previous investigations on the gas-phase ion chemistry of diphosphate species to the [M(1)M(2)HP(2)O(7)](-) ions where M(1) and M(2) are the same or different and correspond to the Li, Na, K, Cs, and Rb cations. The diphosphate ions are formed by electrospray ionization of 10(-4) M solutions of Na(5)P(3)O(10) in CH(3)CN/H(2)O (1/1) and MOH bases or M salts as a source of M(+) cations. The joint application of mass spectrometric techniques and quantum-mechanical calculations makes it possible to characterize the gaseous [M(1)M(2)HP(2)O(7)](-) ions as a mixed ionic population formed by two isomeric species: linear diphosphate anion coordinated to two M(+) cations (group I) and [PO(3)M(1)M(2)HPO(4)](-) clusters (group II). The relative gas-phase stabilities and activation barriers for the isomerization I-->II, which depend on the nature of the M(+) cations, highlight the electronic susceptibility of P-O-P bond breaking in the active site of enzymes. The previously unexplored gas-phase reactivity of [M(1)M(2)HP(2)O(7)](-) ions towards alcohols of different acidity was investigated by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). The reaction proceeds by addition of the alcohol molecule followed by elimination of a water molecule.

Anions↗
PubMed2001

Ypt protein prenylation depends on the interplay among levels of Rab escort protein and geranylgeranyl diphosphate in yeast cells.

Farnesyl diphosphate (FPP), an intermediate of the sterol biosynthetic pathway, is used by farnesyl transferase to farnesylate, among others, the Ras proteins, and by geranylgeranyl diphosphate synthase to produce geranylgeranyl diphosphate (GGPP). GGPP is then transferred by geranylgeranyl transferase II (GGTase II) to Rab/Ypt members of the Ras superfamily known to be required at all stages of vesicle transport in both mammals and yeast. Formation of a complex between a Rab/Ypt protein and an accessory protein named the Rab escort protein (REP) is a prerequisite for GGTase II substrate recognition. Little is known about the factors that regulate GGTase II activity in living cells but, based on available data, it seems possible that vesicle transport in higher eukaryotes is regulated by the levels of prenylated Rab/Ypt proteins in the cells. Here we show that the levels of REP play an important role in regulating GGTase II activity in yeast cells if sufficient substrates are present. Moreover, overexpression of REP causes, directly or indirectly, an increased level of Ypt substrates available for prenylation, which in turn leads to the depletion of the GGPP pool in the cell. Overall our data suggest that the levels of REP and the availability of GGPP play a role in regulating Ypt protein prenylation.

Alkyl and Aryl Transferases↗
PubMed1996

Cloning, characterization, and heterologous expression of cDNAs for farnesyl diphosphate synthase from the guayule rubber plant reveals that this prenyltransferase occurs in rubber particles.

Two farnesyl diphosphate synthase (FPS) cDNA's from a guayule stembark library were isolated and characterized. Both encode M(r) 39,000 proteins containing 432 amino acids that differ slightly in their deduced molecular weights and isoelectric points. They both contain the DDXXD motifs that are characteristic of prenyltransferases, and both isoforms show high homology to other plant FPS sequences but less overall homology to FPS sequences from nonplant sources. The two isoforms differ by 5% in their amino acid sequence. When expressed in Escherichia coli, each guayule isoform exhibits high specific activity that produces farnesyl diphosphate as the major isoprenoid and small amounts of geranyl diphosphate. Biochemical and immunological evidence also indicates that FPS is associated with guayule rubber particles. Antibodies to chicken FPS cross-react with both guayule isoforms expressed in E. coli and recognize a low abundance M(r) 39,000 protein in rubber particles purified from guayule stembark. Guayule FPS sequences show high homology to peptide fragments of the prenyltransferase associated with rubber particles from Hevea brasiliensis, suggesting that this enzyme may be important for rubber biosynthesis in both species.

Alkyl and Aryl Transferases↗
PubMed1976

Purification, some properties and quaternary structure of the D-ribulose 1,5-diphosphate carboxylase of Alcaligenes eutrophus.

D-Ribulose 1,5-diphosphate carboxylase has been purified from autotrophically grown cells of the facultative chemolithotrophic hydrogen bacterium Alcaligenes eutrophus. The enzyme was homogeneous by the criteria of polyacrylamide gel electrophoresis. The molecular weight of the enzyme was 505000 determined by gel filtration and sucrose density gradient centrifugation, and a sedimentation coefficient of 18.2 S was obtained. It was demonstrated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis that the enzyme consists of two types of subunits of molecular weight 52000 and 13000. Electron microscopy on the intact and the partially dissociated enzyme lead to the construction of a model for the quaternary structure of the enzyme which is composed of 8 large and 8 small subunits. The most probable symmetry of the enzyme molecule is 4:2:2. Michaelis constant (Km) values for ribulose 1,5-diphosphate, Mg2+, and CO2 were 0.59 mM, 0.33 mM, and 0.066 mM measured under air. Oxygen was a competitive inhibitor with respect to CO2 suggesting that the enzyme also exhibits an oxygenase activity. The oxygenolytic cleavage of ribulose 1,5-diphosphate was shown and a 1:1 stoichiometry between oxygen consumption and 3-phosphoglycerate formation observed.

Alcaligenes↗
PubMed2002

Characterization, heterologous expression and functional analysis of mevalonate diphosphate decarboxylase gene (MVD) of Candida albicans.

Mevalonate diphosphate decarboxylase (MVD) catalyzes the conversion of mevalonate diphosphate to isopentenyl diphosphate, a key building block for a large family of functionally important biomolecules. We have cloned the gene encoding MVD from Candida albicans, and report the first characterization of such a gene from an opportunistic fungal pathogen. Sequence analysis revealed that the MVD comprises 362 amino acid residues with a predicted molecular weight of 39.5 kDa, sharing minimal identity with the human analogue. Analysis of the genomic sequence indicated that the coding frame is interrupted by a small intron of 51 bp. Southern analysis of genomic DNA demonstrated that MVD is a single-copy gene. Furthermore, Southern analysis of electrophoretic karyotypes of C. albicans obtained by pulsed-field gel electrophoresis showed that MVD is located on chromosome 1. Northern analysis revealed that the level of MVD expression is affected by (1) the carbon source in the growth medium, (2) the growth phase, and (3) the growth form of the fungus (yeast-like or hyphal). To demonstrate the biological function of C. albicans MVD, complementation experiments were carried out with an S. cerevisiae strain (erg19(ts)) that is temperature-sensitive for MVD activity. A single copy of the C. albicans MVD gene, under the control of the NOP1 promoter, was able fully to complement the erg19(ts) phenotype, and expression of the epitope-tagged C. albicans MVD was detectable by Western analysis. Furthermore, the low degree of sequence identity between C. albicans MVD and its human analogue raises the possibility that fungal-specific inhibitors can be developed for the enzyme. Thus, C. albicans MVD appears to be an interesting candidate that could be targeted for the development of anti-fungal agents.

Amino Acid Sequence↗