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PubMed1991

On the mechanism of nucleotide diphosphate activation of the ATP-sensitive K+ channel in ventricular cell of guinea-pig.

1. Effects of intracellular nucleotide diphosphates (NDPs) on the ATP-sensitive K+ channel (K+ATP channel) were examined in ventricular cells of guinea-pig heart, using the inside-out patch clamp technique. On formation of inside-out patches in the ATP-free internal solution, the K+ATP channel appeared and then ran down spontaneously. This run-down of the K+ATP channel activity was probably due to dephosphorylation. 2. Millimolar concentrations of various NDPs, e.g. UDP (uridine diphosphate), IDP (inosine diphosphate), CDP (cytidine diphosphate) and GDP (guanosine diphosphate), applied to the internal side of the patch membrane, induced openings of the K+ATP channel after run-down, i.e. in the dephosphorylated state. ADP opened the channel weakly at low concentrations (100 microM) but inhibited it at higher concentrations (1-10 mM). 3. NDP-induced openings of the channel were Mg2+ dependent and inhibited by ATP (100 microM) and glibenclamide (1 microM). None of nucleosides, nucleotide monophosphates nor nucleotide triphosphates induced openings of the channel. Thus, the K+ATP channel may have a Mg(2+)-dependent NDP-binding site, which induces openings of the dephosphorylated channel in ATP-free solution, in addition to the Mg(2+)-independent ATP-binding inactivation site and phosphorylation site. 4. In inside-out patches, pinacidil (a K+ATP channel opener) activated the K+ATP channel in the phosphorylated state but not in the dephosphorylated state. In the presence of NDPs (UDP, IDP, CDP, GDP), however, pinacidil (30 microM) enhanced openings of the dephosphorylated K+ATP channel prominently. 5. From the above results, we concluded that NDP-binding to the specific site has similar effects to channel phosphorylation, i.e. it keeps the K+ATP channel in an operative state in ATP-free solution and enhances the pinacidil-induced channel openings.

Adenosine Diphosphate↗
PubMed1999

Inhibition of nucleoside diphosphate kinase in rat liver mitochondria by added 3'-azido-3'-deoxythymidine.

The effect of 3'-azido-3'-deoxythymidine on nucleoside diphosphate kinase of isolated rat liver mitochondria has been studied. This is done by monitoring the increase in the rate of oxygen uptake by nucleoside diphosphate (TDP, UDP, CDP or GDP) addition to mitochondria in state 4. It is shown that 3'-azido-3'-deoxythymidine inhibits the mitochondrial nucleoside diphosphate kinase in a competitive manner, with a Ki value of about 10 microM as measured for each tested nucleoside diphosphate. It is also shown that high concentrations of GDP prevent 3'-azido-3'-deoxythymidine inhibition of the nucleoside diphosphate kinase.

Animals↗
PubMed2005

Identification and active expression of the Mycobacterium tuberculosis gene encoding 5-phospho-{alpha}-d-ribose-1-diphosphate: decaprenyl-phosphate 5-phosphoribosyltransferase, the first enzyme committed to decaprenylphosphoryl-d-arabinose synthesis.

Decaprenylphosphoryl-d-arabinose, the lipid donor of mycobacterial d-arabinofuranosyl residues, is synthesized from phosphoribose diphosphate rather than from a sugar nucleotide. The first committed step in the process is the transfer of a 5-phosphoribosyl residue from phosphoribose diphosphate to decaprenyl phosphate to form decaprenylphosphoryl-5-phosphoribose via a 5-phospho-alpha-d-ribose-1-diphosphate:decaprenyl-phosphate 5-phospho-ribosyltransferase. A candidate for the gene encoding this enzyme (Rv3806c) was identified in Mycobacterium tuberculosis, primarily via its homology to one of four genes responsible for d-arabinosylation of nodulation factor in Azorhizobium caulinodans. The resulting protein was predicted to contain eight or nine transmembrane domains. The gene was expressed in Escherichia coli, and membranes from the expression strain of E. coli but not from a control strain of E. coli were shown to convert phosphoribose diphosphate and decaprenyl phosphate into decaprenylphosphoryl-5-phosphoribose. Neither UDP-galactose nor GDP-mannose was active as a sugar donor. The enzyme favored polyprenyl phosphate with 50-60 carbon atoms, was unable to use C-20 polyprenyl phosphate, and used C-75 polyprenyl phosphate less efficiently than C-50 or C-60. It requires CHAPS detergent and Mg(2+) for activity. The Rv3806c gene encoding 5-phospho-alpha-d-ribose-1-diphosphate:decaprenyl-phosphate 5-phosphoribosyltransferase is known to be essential for the growth of M. tuberculosis, and the tuberculosis drug ethambutol inhibits other steps in arabinan biosynthesis. Thus the Rv3806c-encoded enzyme appears to be a good target for the development of new tuberculosis drugs.

Antitubercular Agents↗
PubMed2000

Molecular cloning of an Atlantic salmon nucleoside diphosphate kinase cDNA and its pattern of expression during embryogenesis.

To gain insight into the process of development in Atlantic salmon (Salmo salar), we sought to identify genes that were differentially expressed at gastrulation. A polymerase chain reaction-based differential screening strategy allowed for the isolation of an Atlantic salmon nucleoside diphosphate kinase cDNA (nm23). Structural characterisation showed a high degree of homology with a large number of previously isolated nucleoside diphosphate kinases (NM23s), both prokaryote and eukaryote, though it represents the first teleost nucleoside diphosphate kinase identified. Highest similarities were found with the type 1 and type 2 NM23 isoforms of mammals. Phylogenetic analysis indicates that the duplication event that gave rise to these isoforms occurred after the splitting of tetrapods and fish, suggesting that the salmon NM23 represents a more ancestral isoform. The position of the salmon sequence on the phylogenetic tree indicates that the salmon genome is expected to have at least three copies of genes from the nm23 gene family. Northern blot analysis showed a single transcript of approximately 0.7 kb in both embryonic and adult tissues. Examination of the temporal pattern of expression of salmon nucleoside diphosphate kinase during embryonic development revealed that this gene is first expressed at the time of gastrulation. Nucleoside diphosphate kinases are thought to have a vital role in regulatory processes such as signal transduction, proliferation and differentiation. Taken together, these results suggest that nucleoside diphosphate kinases have an important role to play in early embryogenic development in vertebrates.

Amino Acid Sequence↗
PubMed2006

Structure-activity relationships of uridine 5'-diphosphate analogues at the human P2Y6 receptor.

The structure-activity relationships and molecular modeling of the uracil nucleotide activated P2Y6 receptor have been studied. Uridine 5'-diphosphate (UDP) analogues bearing substitutions of the ribose moiety, the uracil ring, and the diphosphate group were synthesized and assayed for activity at the human P2Y6 receptor. The uracil ring was modified at the 4 position, with the synthesis of 4-substituted-thiouridine 5'-diphosphate analogues, as well as at positions 2, 3, and 5. The effect of modifications at the level of the phosphate chain was studied by preparing a cyclic 3',5'-diphosphate analogue, a 3'-diphosphate analogue, and several dinucleotide diphosphates. 5-Iodo-UDP 32 (EC50 = 0.15 microM) was equipotent to UDP, while substitutions of the 2'-hydroxyl (amino, azido) greatly reduce potency. The 2- and 4-thio analogues, 20 and 21, respectively, were also relatively potent in comparison to UDP. However, most other modifications greatly reduced potency. Molecular modeling indicates that the beta-phosphate of 5'-UDP and analogues is essential for the establishment of electrostatic interactions with two of the three conserved cationic residues of the receptor. Among 4-thioether derivatives, a 4-ethylthio analogue 23 displayed an EC50 of 0.28 microM, indicative of favorable interactions predicted for a small 4-alkylthio moiety with the aromatic ring of Y33 in TM1. The activity of analogue 19 in which the ribose was substituted with a 2-oxabicyclohexane ring in a rigid (S)-conformation (P = 126 degrees , 1'-exo) was consistent with molecular modeling. These results provide a better understanding of molecular recognition at the P2Y6 receptor and will be helpful in designing selective and potent P2Y6 receptor ligands.

Cell Line, Tumor↗
PubMed1990

Stimulation of cyclic GMP production in cultured endothelial cells of the pig by bradykinin, adenosine diphosphate, calcium ionophore A23187 and nitric oxide.

1. The effects of bradykinin, adenosine diphosphate, calcium ionophore A23187 and nitric oxide on the production of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and guanosine 3':5'-cyclic monophosphate (cyclic GMP) were investigated in cultured aortic endothelial cells of the pig. 2. Bradykinin (10(-7) M), adenosine diphosphate (3 x 10(-5) M), nitric oxide (2 x 10(-6) M) and A23187 (10(-6) M) stimulated the production of cyclic GMP. This stimulation reached a maximum within 1 min and declined rapidly with the first three agonists whereas that induced by A23187 was long-lasting. 3. These concentrations of bradykinin, A23187 and nitric oxide had no effect on cyclic AMP production. However, adenosine diphosphate (3 x 10(-5) M) slightly but significantly enhanced its production by about 1.7 fold. 4. The basal content of cyclic GMP in endothelial cells was significantly reduced by haemoglobin (10(-5) M, a scavenger of endothelium-derived relaxing factor(s] and methylene blue (10(-5) M, an inhibitor of the activation of soluble guanylate cyclase) and was significantly enhanced by superoxide dismutase (500 u ml-1, a scavenger of superoxide anions). The basal content of cyclic GMP was not affected by NG-monomethyl-L-arginine (10(-5) M, a specific inhibitor of the formation of nitric oxide from L-arginine) and was slightly but significantly increased by its D-enantiomer, NG-monomethyl-D-arginine. 5. The production of cyclic GMP stimulated by bradykinin, adenosine diphosphate, A23187 and nitric oxide was inhibited by haemoglobin (10 5M) and methylene blue (10- M) but was unaffected by superoxide dismutase (500 u ml 1)- 6. The production of cyclic GMP stimulated by bradykinin, adenosine diphosphate or A23187, but not that stimulated by nitric oxide, was significantly reduced by N0-monomethyl-L-arginine (10-M). The production of cyclic GMP evoked by nitric oxide, but not that induced by the other three agents, was enhanced significantly by N0-monomethyl-D-arginine by about 1.5 fold. 7. These data indicate that the endothelium-dependent vasodilators bradykinin, adenosine diphosphate and A23187 activate the production of cyclic GMP in endothelial cells via the synthesis of nitric oxide, which in turn stimulates the soluble guanylate cyclase.

Adenosine Diphosphate↗
PubMed1989

Effect of uridine 5'-diphosphate on cryogenic brain edema in rabbits.

This study was undertaken to examine the effect of uridine 5'-diphosphate, administered intravenously or intraperitoneally, on cold injury-induced brain edema in rabbits. Bolus injection or continuous intravenous infusion of uridine 5'-diphosphate 26 hours after a lesion was established had adverse effects, such as increased intracranial pressure and lowered systolic arterial blood pressure and cerebral perfusion pressure for approximately 10-29 minutes, but these parameters did not change appreciably from 29 minutes to 3 hours after administration. Intraperitoneally administered uridine 5'-diphosphate did not affect these parameters appreciably during 3 hours. Thus, the intravenous administration of uridine 5'-diphosphate is harmful under neurosurgical conditions. In contrast, 10 mg/kg/day i.p. uridine 5'-diphosphate pretreatment and posttreatment, beginning 24 hours before and continuing until 24 hours after the insult, significantly reduced neurologic abnormalities, Evans blue extravasation, water content in the injured gray matter, and intracranial pressure without affecting water content in the white matter. Intravenous dexamethasone pretreatment and posttreatment in this setting significantly reduced only neurologic abnormalities. However, there were no significant differences between intraperitoneal uridine 5'-diphosphate and intravenous dexamethasone effects on cold-injured brain.

Animals↗
PubMed1975

Ribulose diphosphate carboxylase/oxygenase. IV. Regulation by phosphate esters.

The stimulation or inhibition of ribulose diphosphate oxygenase by a variety of compounds is compared with the reported effects on these compounds on the ribulose diphosphate carboxylase activity. A possible transition state analog of ribulose diphosphate, 2-carboxyribitol 1, 5-diphosphate, at a molar ratio of inhibitor to enzyme of 10 to 1, irreversibly inactivates the oxygenase and carboxylase activities. This is consistent with the hypothesis that there may be a single active site for both the carboxylase and oxygenase activities. Several compounds of the reductive pentose photosynthetic carbon cycle act as effectors of the ribulose diphosphate oxygenase in a manner complementary to their reported effect upon the carboxylase. Ribose 5-phosphate inhibits the oxygenase with an apparent Ki of 1.8 mM, but it is reported to activate the carboxylase; fructose 6-phosphate and glucose 6-phosphate act similarly but are less effective than ribose 5-phosphate. Fructose 1. 6-diphosphate stimulates the oxygenase at low magnesium ion concentrations. The stimulatory effect of 6-phosphogluconate on the oxygenase is associated with a 3-fold reduction of the Km (Mg2+). ATP inhibits the oxygenase but has been reported to stimulate the carboxylase; pyrophosphate acts in an opposite manner. From these results it appears that the ratio of carboxylase to oxygenase activity may be a variable factor with predictable subsequent alteration in the ratio between photosynthetic CO2 fixation and photorespiration.

Adenosine Triphosphate↗
PubMedDate not supplied

[Covalent binding of 1,N(6)-ethenoadenosine diphosphate to catalytic and noncatalytic sites of chloroplast ATP-synthase].

The catalytic and noncatalytic sites of the chloroplast coupling factor (CF1) were selectively modified by incubation with the dialdehyde derivative of the fluorescent adenosine diphosphate analogue 1,N(6)-ethenoadenosine diphosphate. The modified CF1 was reconstituted with EDTA-treated chloroplast thylakoid membranes. The influence of light-induced transmembrane proton gradient and of phosphate ions on the fluorescence of 1,N(6)-ethenoadenosine diphosphate covalently bound to catalytic sites of reconstituted CF1 (ATP-synthase) was studied. Upon illumination of thylakoid membranes with saturating white light, the quenching of fluorescence of covalently bound 1,N(6)-ethenoadenosine diphosphate was observed. The quenching was reversed by the addition of inorganic phosphate to the reaction mixture in the dark. Repeated illumination induced the quenching once again: however, the addition of phosphate ions did not affect the fluorescence intensity now. When 1,N(6)-ethenoadenosine diphosphate was covalently bound to noncatalytic sites of ATP-synthase, no similar fluorescent changes were observed. The interrelation of the observed changes of 1,N(6)-ethenoadenosine diphosphate fluorescence and the mechanism of energy-dependent changes in the structure of the catalytic site of ATP-synthase is discussed.

Adenosine Diphosphate↗
PubMed1995

Temporal and spatial distributions of yeast nucleoside diphosphate kinase activities and its association with the Cdc8p.

Nucleoside diphosphate kinase (E.C. 2.7.4.6.) is a broad substrate-specific enzyme that catalyzes the phosphorylation of nucleoside diphosphates to the corresponding triphosphates in nucleic acid biosynthesis. In this report, we investigate its spatial and temporal distributions in yeast to understand how the enzyme exerts its gene function(s). Our results show that the enzyme is predominantly cytoplasmic. A substantial amount of enzyme activity (40-50%) may be associated with the cell membrane. Less than 1% of total activity was detected in the nuclear fraction. Approximately 3% was found in the mitochondrial fraction. When yeast cultures were synchronized, we found that Saccharomyces cerevisiae nucleoside diphosphate kinase did not show cell cycle periodicity, as Schizosaccharomyces pombe enzyme did. To explore its link with DNA synthesis, we investigated its relationship with the Cdc8p (dTMP kinase). We demonstrated a physical interaction between these proteins in vitro, as evidenced that the GST:Cdc8p protein affinity column could retain a subpopulation of nucleoside diphosphate kinase activity from yeast crude extract. Furthermore, when GST:Cdc8p protein was expressed in yeast, the protein could bind to the glutathione-agarose, along with nucleoside diphosphate kinase, suggesting that there is an interaction between GST:Cdc8p and nucleoside diphosphate kinase in vivo. Our results provide evidence for at least a two-enzyme complex that may well facilitate nucleotide channeling in the cell.

Cell Cycle↗
PubMed1979

Nucleotide release from tubulin and nucleoside-5'-diphosphate kinase action in microtubule assembly.

ATP and UTP support microtubule assembly through the action of brain nucleoside-5'-diphosphate kinase on GDP. Penningroth and Kirschner (1977) J. Mol. Biol. 115, 643-673) have proposed that microtubule assembly may occur by either of two mechanisms: indirectly, through nucleoside-5'-diphosphate kinase-catalyzed phosphorylation of uncomplexed GDP and directly by nucleoside-5'-diphosphate kinase-mediated transphosphorylation of tubulin-bound GDP at low tubulin concentrations. We find the rates of GDP and GTP release (0.68 and 0.32 min-1, respectively) are sufficiently fast relative to assembly to permit GDP release, phosphorylation, and GTP binding as the sole mechanism of nucleoside-5'-diphosphate kinase action in microtubule assembly. Computer simulation studies accord with the conclusion that GDP release is rapid relative to microtubule assembly. The specific activity of the nucleoside-5'-diphosphate kinase is 1.7 nmol/min/mg of microtubular protein under the conditions studied. Pulse-chase experiments with tubulin . [14C]GDP complex and the rapidity of GDP phosphorylation by the kinase are in agreement with this scheme. Finally, it was observed that the extent and rate of microtubule assembly depends upon the [ATP]/[ADP] ratio.

Adenosine Diphosphate↗
PubMed1990

Menadiol diphosphate, a new substrate for non-specific alkaline phosphatase in histochemistry and immunohistochemistry.

Menadiol diphosphate was introduced as a new substrate for nonspecific alkaline phosphatase, following a search for new and less expensive substrates, which give a more sensitive response and are easily synthesized in the laboratory. Menadiol released by phosphatase action can be assayed by its reduction of tetrazolium salts, or it can be coupled with diazonium salts; alternatively, the phosphate can be trapped by metal ions. The synthesis and purification of menadiol diphosphate are described, and it was shown to be sufficiently stable for qualitative and semiquantitative histochemistry, as well as for the immunohistochemistry of enzymes and cytoskeletal proteins with nonspecific alkaline phosphatase as the enzyme label. For qualitative as well as semiquantitative histochemistry and immunohistochemistry, the best results were obtained by applying the method with nitro-blue tetrazolium (NBT) to acetone-chloroform pretreated cryostat sections. Tetranitro-blue tetrazolium (TNBT), benzothiazolylphthalhydrazidyl tetrazolium (BSPT) and various diazonium salts were less suitable. Fast Blue BB and VB produced satisfactory results. Ce3+ ions and the DAB-Ni-H2O2 procedure yielded better results than Ca2+ ions in the Co-(NH4)2S visualization method. The NBT method with menadiol diphosphate is superior to existing methods employing azo, azoindoxyl or tetrazolium salts and to metal precipitation methods. The Ce3+ technique and the NBT/menadiol diphosphate method give similar results, and appear to be of equal value. In qualitative histochemistry and immunohistochemistry the NBT/menadiol diphosphate method resulted in higher quantities of precisely localized stain. Semiquantitative histochemistry with minimal incubation revealed more favorable kinetics for the menadiol diphosphate method, especially when using NBT.

Alkaline Phosphatase↗
PubMed1975

Regulation of glucose-6-phosphate dehydrogenase in spinach chloroplasts by ribulose 1,5-diphosphate and NADPH/NADP+ ratios.

The activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) FROM SPINACH CHLOROPLASTS IS STRONGLY REGULATED BY THE RATIO OF NADPH/NADP+, with the extent of this regulation controlled by the concentration of ribulose 1,5-diphosphate. Other metabolites of the reductive pentose phosphate cycle are far less effective in mediating the regulation of the enzyme activity by NADPH/NADP+ ratio. With a ratio of NADPH/NADP+ of 2, and a concentration of ribulose 1,5-diphosphate of 0.6 mM, the activity of the enzyme is completely inhibited. This level of ribulose 1,5-diphosphate is well within the concentration range which has been reported for unicellular green algae photosynthesizing in vivo. Ratios of NADPH/NADP+ of 2.0 have been measured for isolated spinach chloroplasts in the light and under physiological conditions. Since ribulose 1,5-diphosphate is a metabolite unique to the reductive pentose phosphate cycle and inhibits glucose-6-phosphate dehydrogenase in the presence of NADPH/NADP+ ratios found in chloroplasts in the light, it is proposed that regulation of the oxidative pentose phosphate cycle is accomplished in vivo by the levels of ribulose 1,5-diphosphate, NADPH, and NADP+. It already has been shown that several key reactions of the reductive pentose phosphate cycle in chloroplasts are regulated by levels of NADPH/NADP+ or other electron-carrying cofactors, and at least one key-regulated step, the carboxylation reaction is strongly affected by 6-phosphogluconate, the metabolic unique to the oxidative pentose phosphate cycle. Thus there is an interesting inverse regulation system in chloroplasts, in which reduced/oxidized coenzymes provide a general regulatory mechanism. The reductive cycle is activated at high NADPH/NADP+ ratios where the oxidative cycle is inhibited, and ribulose 1,5-diphosphate and 6-phosphogluconate provide further control of the cycles, each regulating the cycle in which it is not a metabolite.

Adenosine Triphosphate↗
PubMed1989

Kinetic studies on the prenyl chain elongation by undecaprenyl diphosphate synthase with artificial substrate homologues.

In the undecaprenyl diphosphate synthase reaction, an allylic substrate homologue, (2Z,6E,10E)-4-methyl-geranylgeranyl diphosphate was found to be a potent competitive inhibitor against the allylic primer, (2Z,6E,10E)-geranylgeranyl diphosphate. On the other hand, it acted as a strong noncompetitive inhibitor against isopentenyl diphosphate. On the basis of these facts, the topology of the substrate-binding sites as well as the reason why the synthase reaction with (E)-3-methyl-3-pentenyl diphosphate always stops completely at the first stage of condensation, yielding an allylic diphosphate with a methyl group at the 4-position, are discussed.

Alkyl and Aryl Transferases↗
PubMed1975

"Single Addition" substrates for the synthesis of specific oligoribonucleotides with polynucleotide phosphorylase. Synthesis of 2'-(alpha-methoxyethy) nucleoside 5'-diphosphates.

A number of synthetic methods for the preparation of the 2-O-(alpha-methoxyethyl) derivatives of the 5-diphosphates of adenosine, cytidine, guanosine, and uridine have been studied in order to provide nucleotide substrates that can be applied to the synthesis of specific oligoribonucleotides using polynucleotide phosphorylase. The reaction of nucleoside 5-diphosphates with methyl vinyl ether for a limited time produces low yields of the corresponding 2-O-(alpha-methoxyethyl) derivatives because the rate of methoxyethylation of the 3-hydroxyl groups. A study of the rates of acidic hydrolysis of alpha-methoxyethyl groups in the 2 and 3 positions of nucleosides and nucleotides has been made, and the results obtained form the basis of a more efficient method for the synthesis of the blocked nucleoside diphosphates. The method involves the reaction of nucleoside 5-diphosphates with methyl vinyl ether to give the corresponding 2,3-di-O-(alpha-methoxyethyl)nucleoside 5-diphosphates, and exploits the fact that, in the acidic hydrolysis of these derivatives, the rate of removal of the 3-methoxyethyl group is about twice that of the group in the 2 position. Alternative syntheses were based on the phosphorylation of methoxyethylated nucleosides and nucleotides. The derivatives, 2-O- and 2,3-di-O-(alpha-methoxyethyl)uridine, were prepared by the methoxyethylation of 3,5-di-O-acetyluridine and 5-O-acetyluridine followed by removal of the acetyl groups. The corresponding guanosine derivatives were made by the synthetic routes: (i) guanosine leads to O-2,O-3,O-5,N-2-tetrabenzoylguanosine leads to 2-N-benzoylguanosine leads to O3-acetyl-N-2,O5-dibenzoylguanosine leads to 2-O-(alpha-methoxyethyl)guanosine, and (ii) 2,3-O-isopropylideneguanosine leads to N-2,O5-diacetyl-2,3-O-isopropylideneguanosine leads to N-2,O-5-diacetylguanosine leads to 2,3-di-O-(alpha-methoxyethyl)guanosine. These methoxyethylated nucleosides were converted to the corresponding 5-phosphates by reaction with cyanoethyl phosphate and dicyclohexylcarbodiimide, and then to the corresponding 5-diphosphates by subsequent reaction with 1,1-carbonyldiimidazole and inorganic phosphate.

Chromatography, DEAE-Cellulose↗
PubMed1996

Identification of significant residues in the substrate binding site of Bacillus stearothermophilus farnesyl diphosphate synthase.

Farnesyl diphosphate synthases have been shown to possess seven highly conserved regions (I-VII) in their amino acid sequences [Koyama et al. (1993) J. Biochem. (Tokyo) 113, 355-363]. Site-directed mutants of farnesyl diphosphate synthase from Bacillus stearothermophilus were made to evaluate the roles of the conserved aspartic acids in region VI and lysines in regions I, V, and VI. The aspartate at position 224 was changed to alanine or glutamate (mutants designated as D224A and D224E, respectively); aspartates at positions 225 and 228 were changed to isoleucine and alanine (D225I, D228A); lysine at position 238 was changed to either alanine or arginine (K238A, K238R). The lysines at positions 47 and 183 were changed to isoleucine and alanine (K471, K183A), respectively. Kinetic analyses of the wild-type and mutant enzymes indicated that the mutagenesis of Asp-224 and Asp-225 resulted in a decrease of Kcat values of approximately 10(4)- to 10(5)-fold compared to the wild type. On the other hand, D228A showed a Kcat value approximately one-tenth of that of the wild type, and the k(m) value for isopentenyl diphosphate increased approximately 10-fold. Both K471 and K183A showed k(m) values for isopentenyl diphosphate 20-fold larger and kcat values 70-fold smaller than the wild type. These results suggest that the two conserved lysines in regions I and V contribute to the binding of isopentenyl diphosphate and that the first and the second aspartates in region VI are involved in catalytic function. Aspartate-228 is also important for the binding of isopentenyl diphosphate rather than for catalytic reaction.

Acyclic Monoterpenes↗
PubMed1998

Two subunits of heptaprenyl diphosphate synthase of Bacillus subtilis form a catalytically active complex.

Heptaprenyl diphosphate synthase of Bacillus subtilis, which participates in the biosynthesis of the side chain of menaquinone-7, is composed of two dissociable subunits, component I and component II, which are encoded by two cistrons in a novel gene cluster of gerC operon [Zhang, Y.-W., et al. (1997) J. Bacteriol. 179, 1417-1419]. This enzyme essentially requires the coexistence of both subunits for its catalysis. Expression vector systems for the two structural genes, gerC1 and gerC3, were constructed separately, and the two components were overproduced in Escherichia coli cells. After purification, their dynamic interactions in forming a catalytically active complex were investigated by gel filtration and immunoblotting analyses. When a mixture of the two components that had been preincubated in the presence of Mg2+ and farnesyl diphosphate was subjected to Superdex 200 gel filtration, a significant elution peak appeared in a region earlier than those observed when they were chromatographed individually. This fraction contained both components I and II, and it corresponded to a molecular mass that is in accord with the sum of the values of the two components. Cross-linking studies indicate that the two essential subunits, farnesyl diphosphate, and Mg2+ form a ternary complex which seems to represent a catalytically active state of the heptaprenyl diphosphate synthase. On the other hand, no complex was formed in the presence of isopentenyl diphosphate or inorganic pyrophosphate and Mg2+. A photoaffinity analogue of farnesyl diphosphate was shown to preferentially label the component I protein, suggesting that component I possesses a specific affinity for the allylic substrate. Furthermore, the photoaffinity labeling of component I significantly increased in the presence of component II. The mechanism of catalysis of this unique heteromeric enzyme is understood by assuming that association and dissociation of the two subunits facilitate turnover of catalysis for the synthesis of the amphipathic product from soluble substrates.

Alkyl and Aryl Transferases↗
PubMed2006

Escherichia coli type I isopentenyl diphosphate isomerase: structural and catalytic roles for divalent metals.

Isopentenyl diphosphate isomerase (IDI) catalyzes the essential conversion of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) in the mevalonate entry into the isoprenoid biosynthetic pathway. Two convergently evolved forms of IDI are known. Type I IDI, which is found in Eukarya and many Bacteria, catalyzes the isomerization of IPP and DMAPP by a protonation-deprotonation mechanism. The enzyme requires two divalent metal ions for activity. An X-ray structure of type I IDI from crystals soaked with (N,N-dimethylamino)-1-ethyl diphosphate (NIPP), a potent transition-state analogue for the carbocationic intermediate in the isomerization reaction, shows one of the metals in a His(3)Glu(2) hexacoordinate binding site, while the other forms a bridge between the diphosphate moiety of the substrate and the enzyme (Wouters, J.; et al. J. Biol. Chem. 2003, 278, 11903). Reconstitution of metal-free recombinant Escherichia coli type I IDI with several divalent metals-Mg(2+), Mn(2+), Zn(2+), Co(2+), Ni(2+), and Cd(2+)-generated active enzyme. Freshly purified IDI contained substoichiometric levels of a single metal ion, presumably bound in the hexacoordinate site. When NIPP was added to the disruption and purification buffers of enzyme, the purified protein contained 0.72 equiv of Mg(2+), 0.92 equiv of Zn(2+), and 0.10 equiv of Mn(2+). These results are consistent with a structure in which Mg(2+) facilitates diphosphate binding and Zn(2+) or Mn(2+) occupies the hexacoordinate site.

Buffers↗