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PubMed1999

Identification of the GGPS1 genes encoding geranylgeranyl diphosphate synthases from mouse and human.

E,E,E-Geranylgeranyl diphosphate (GGPP) is an important precursor of carotenoids and geranylgeranylated proteins such as small G proteins. In this study, we have identified mouse and human GGPP synthase genes. Sequence analysis showed that mouse and human GGPP synthases share a high level of amino acid identity (94%) with each other, and share a high level of similarity (45-50%) with GGPP synthases of lower eukaryotes, but only weak similarity (22-31%) to plant and prokaryotic GGPP synthases. Both of the newly identified GGPP synthase genes from mouse and human were expressed in Escherichia coli, and their gene products displayed GGPP synthase activity when isopentenyl diphosphate and farnesyl diphosphate were used as substrates. The GGPP synthase activity of these genes was also confirmed by demonstrating carotenoid synthesis after co-transformation of E. coli with a plasmid expressing the crt genes derived from Erwinia uredovora, and a plasmid expressing either the mouse or human GGPS1 gene. Southern blot analysis suggests that the human GGPS1 gene is a single copy gene.

Alkyl and Aryl Transferases↗
PubMed1993

Isoprenoid diphosphate utilization by recombinant human farnesyl:protein transferase: interactive binding between substrates and a preferred kinetic pathway.

The catalytic utilization of dimethylallyl, geranyl, farnesyl, and geranylgeranyl diphosphates in the reaction catalyzed by recombinant human farnesyl:protein transferase (hFPTase) has been examined in the presence of three different protein substrates, Ras-CVLS, Ras-CVIM, and Ras-CAIL. hFPTase catalyzed both farnesylation and geranylation of Ras-CVLS and of Ras-CVIM but not of Ras-CAIL. Geranylgeranylation was observed, but only when Ras-CVIM was the acceptor substrate. Steady-state initial velocity and dead-end inhibitor studies indicate that hFPTase-catalyzed geranylation, like bovine FPTase-catalyzed farnesylation, proceeds through a random order, sequential mechanism. Surprisingly, however, Michaelis constants for a given protein acceptor substrate varied depending upon which isoprenoid diphosphate was used as the donor substrate, showing that these substrates do not bind independently to the enzyme (under catalytic conditions). In addition, at very high concentrations of Ras-CVIM, substrate inhibition was observed in the presence of both FPP and GPP. Isotope partitioning studies showed that, at high concentrations of Ras-CVIM, more than 80% of the bound farnesyl diphosphate (FPP) can be trapped as product, suggesting that the binary complex is catalytically competent and that the ternary complex proceeds to product faster than it releases FPP. The release rate of FPP from the binary complex was calculated to be 0.05 s-1, which is only about eight times greater than kcat. Thus, the binding of FPP to the enzyme in the presence of the protein substrate is not an equilibrium situation.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkyl and Aryl Transferases↗
PubMed2000

Spectroscopic detection of transient thiamin diphosphate-bound intermediates on benzoylformate decarboxylase.

Thiamin diphosphate (ThDP)-dependent enzymes catalyze a range of transformations, such as decarboxylation and ligation. We report a novel spectroscopic assay for detection of some of the ThDP-bound intermediates produced on benzoylformate decarboxylase. Benzoylformate decarboxylase was mixed with its alternate substrate p-nitrobenzoylformic acid on a rapid-scan stopped-flow instrument, resulting in formation of three absorbing species (lambda(max) in parentheses): I(1) (a transient at 620 nm), I(2) (a transient at 400 nm), and I(3) (a stable absorbance with lambda(max) > 730 nm). Analysis of the kinetics of the two transient species supports a model in which a noncovalent complex of the substrate and the enzyme is converted to the first covalent intermediate I(1); the absorbance corresponding to I(1) is probably a charge-transfer band arising from the interaction of the thiamin diphosphate-p-nitrobenzoylformic acid covalent adduct (2-p-nitromandelylThDP) and the enzyme. The rate of disappearance of I(1) parallels the rate of formation of I(2). Chemical models suggest the lambda(max) of I(2) (near 400 nm) to be appropriate to the enamine, a key intermediate in ThDP-dependent reactions resulting from the decarboxylation of the thiamin diphosphate-p-nitrobenzoylformic acid covalent adduct. Therefore, the rate of disappearance of I(1) and/or the appearance of I(2) directly measure the rate of decarboxylation. A relaxation kinetic treatment of the pre-steady-state kinetic data also revealed a hitherto unreported facet of the mechanism, alternating active-sites reactivity. Parallel studies of the His70Ala BFD active-site variant indicate that it cannot form the complex reported by the charge-transfer band (I(1)) at the level of the wild-type protein.

Alanine↗
PubMed1994

Crystal structure of recombinant farnesyl diphosphate synthase at 2.6-A resolution.

The synthesis of farnesyl diphosphate (FPP), a key intermediate in the isoprenoid biosynthetic pathway required for the synthesis of cholesterol and in the formation of prenylated proteins, is catalyzed by the enzyme farnesyl diphosphate synthase (FPS). The crystal structure of avian recombinant FPS, the first three-dimensional structure for any prenyltransferase, was determined to 2.6-A resolution. The enzyme exhibits a novel fold composed entirely of alpha-helices joined by connecting loops. The enzyme's most prominent structural feature is the arrangement of 10 core helices around a large central cavity. Two aspartate-rich sequences that are highly conserved among the isoprenyl diphosphate synthase family of prenyltransferases, and are essential for enzymatic activity, were found on opposite walls of this cavity, with the aspartate side chains approximately 12 A apart and facing each other. The location and metal ion binding properties of these sequences suggest that the conserved aspartate residues participate in substrate binding of catalysis.

Alkyl and Aryl Transferases↗
PubMed1994

Structural and functional roles of the cysteine residues of Bacillus stearothermophilus farnesyl diphosphate synthase.

p-(Chloromercuri)benzoic acid inhibited the catalytic activity of Bacillus stearothermophilus farnesyl diphosphate synthase (FPP synthase), which contains only two cysteine residues at positions 73 and 289. In order to explore the role of the cysteine residues, either or both of them were replaced with phenylalanine or serine. Five mutant enzymes, C73F, C73S, C289F, C289S, and C73S-C289S, were overproduced in Escherichia coli and purified to homogeneity. All of them were active as farnesyl diphosphate synthase, showing specific activities comparable to that of the wild-type enzyme. These results indicate that neither of the cysteines is essential for catalytic function. The C73F mutant, however, was very sensitive to heat treatment, while C73S was as highly stable as the wild type. The Km value of C289F for isopentenyl diphosphate is 10 times that of the wild type. The wild-type enzyme was converted into an oxidized form which was separable from the native enzyme on ion-exchange chromatography, and this conversion was accelerated by cupric ions. Similar conversion has previously been reported by several researchers, who found the occurrence of two forms of pig liver FPP synthase and who attributed this phenomenon to the oxidoreduction of sulfhydryl and disulfide groups. However, even the C73S-C289S mutant, which has no cysteine residues, was also converted into an oxidized form as in the case of the wild-type enzyme. These results provide evidence that residues other than cysteine are involved in the conversion of this enzyme into the oxidized form.

Alkyl and Aryl Transferases↗
PubMed1985

Mechanism of inactivation of Escherichia coli ribonucleotide reductase by 2'-chloro-2'-deoxyuridine 5'-diphosphate: evidence for generation of a 2'-deoxy-3'-ketonucleotide via a net 1,2 hydrogen shift.

Sodium borohydride or ethanethiol protects the Escherichia coli ribonucleoside-diphosphate reductase (RDPR) from inactivation by 2'-chloro-2'-deoxyuridine 5'-diphosphate (ClUDP). Incubation of [3'-3H]ClUDP with RDPR in the presence of NaBH4 allowed trapping of [3H]-2'-deoxy-3'-ketouridine 5'-diphosphate. Degradation of the reduced ketone by a combination of enzymatic and chemical methods indicated that the hydrogen originally present in the 3'-position of ClUDP is transferred to the beta-face of the 2'-position of 2'-deoxy-3'-keto-UDP. RDPR therefore catalyzes a net 1,2 hydrogen shift. Incubation of RDPR with ClUDP in the presence of ethanethiol allowed trapping of 2-methylene-3(2H)-furanone, the species responsible for inactivation of RDPR. Trapped 2-[(ethylthio)methyl]-3(2H)-furanone was identical by 1H NMR spectroscopy with material synthesized chemically. Both subunits of the enzyme are covalently radiolabeled in the reaction of RDPR with [5'-3H]ClUDP. Studies with [3'-3H]ClUDP and prereduced RDPR in the absence of a reductant and with oxidized RDPR indicated that the redox-active thiols of the B1 subunit are not involved in inactivation of the enzyme by ClUDP.

Borohydrides↗
PubMed1975

Hydrolysis of ribonucleoside 3'-diphosphates by rye grass 3'-nucleotidase.

Rye grass 3'-nucleotidase has been purified to apparent homogeneity on Sephadex A-25 and CM-cellulose columns and shown to hydrolyze 2'-O-methyladenosine 3'-monophohate and 2'-deoxyadenosine 3'-monophosphate 35.8 and 542 times more slowly than the normal substrate (3'-AMP), verifying the importance of the 2'-beta-OH group of the substrate in the overall hydrolysis process. Although neither was hydrolyzed as rapidly as 3'-AMP, both the 2'-O-methyl and 2'-deoxy analogs acted as competitive inhibitors of the hydrolysis of 3'-AMP (K-m equals 0.12 mM), with apparent K-i's of 0.39 and 0.51 nM, respectively. In order to determine the possible susceptibility of naturally occurring ribonucleoside 3'-diphosphates, such as guanosine tetraphosphate (ppGpp), to 3'-phosphohydrolase activities, the 3'-nucleotidase was also employed in the attempted pyrophosphorolysis of adenosine 3'-diphosphate and guanosine tetraphosphate. Neither adenosine 3'-diphosphate nor guanosine tetraphosphate was degraded at a significant rate by the nucleotidase, relative to the normal substrate.

Adenine Nucleotides↗
PubMed2005

Evidence for dramatic acceleration of a C-H bond ionization rate in thiamin diphosphate enzymes by the protein environment.

The hypothesis that thiamin diphosphate-dependent enzymes achieve a significant fraction of their catalytic rate acceleration by providing a protein environment that helps to stabilize unstable zwitterionic/dipolar intermediates (including the enamine/C2alpha-carbanion present on all such enzymes) was tested experimentally using the intermediate C2alpha-hydroxyethylthiamin diphosphate (HEThDP) with the Escherichia coli pyruvate dehydrogenase complex and its E1 subunit (PDHc-E1). Using pre-steady-state and steady-state methods, it was shown that HEThDP is a substrate for this enzyme after ionization of the C2alpha-H bond. An experiment was then carried out to measure the PDHc-E1 catalyzed pre-steady-state rate constant for the D --> H exchange from the C2alpha position of HEThDP-d(4), as an indicator of the formation of the enamine. Importantly, the enzyme accelerates the rate of ionization of this bond by a factor of 10(7), corresponding to a 10 kcal/mol stabilization of the enamine intermediate by the enzyme. This finding is likely a general feature of thiamin diphosphate enzymes.

Acetylation↗
PubMed2005

Investigation of the functional contributions of invariant serine residues in yeast mevalonate diphosphate decarboxylase.

Alignment of more than 20 deduced sequences for mevalonate diphosphate decarboxylase (MDD) indicates that serines 34, 36, 120,121, 153, and 155 are invariant residues that map within a proposed interdomain active site cleft. To test possible active site roles for these invariant serines, each has been mutated to alanine. S34A exhibits limited solubility and impaired binding of the fluorescent ATP analogue, trinitrophenyl-ATP (TNP-ATP), suggesting that Ser-34 substitution destabilizes proper enzyme folding. All other serine mutants retain structural integrity, as indicated by their ability to bind TNP-ATP at levels comparable to wild-type enzyme. S153A exhibits a 18-fold inflation in K(d) for Mg-ATP, as indicated by competitive displacement of TNP-ATP; the enzyme also is characterized by a 35-fold inflation in K(m) for Mg-ATP. S155A exhibits a 26-fold inflation in K(m) for Mg-ATP, but competitive displacement of TNP-ATP indicates only a 2-fold inflation in K(d) for this substrate. S155A exhibits both a 16-fold inflation in K(m) for mevalonate diphosphate and a 14-fold inflation in K(i(slope)) for the substrate analogue, diphosphoglycolylproline. These observations suggest roles for Ser-153 and Ser-155 in substrate binding. Catalytic consequences of mutating invariant serines 36, 120, 153, and 155 are modest (<8-fold diminution in k(cat)). In contrast, S121A, which exhibits only modest changes in K(d) for Mg-ATP and K(m) for mevalonate diphosphate, is characterized by a >42,000-fold diminution in k(cat), indicating the critical involvement of Ser-121 in reaction catalysis. The selective involvement of the latter of two tandem serine residues (Ser-120, Ser-121) in a conserved sequence motif suggests mechanistic similarities within the GHMP kinase superfamily of proteins.

Adenosine Triphosphate↗
PubMed2000

Intersubunit location of the active site of farnesyl diphosphate synthase: reconstruction of active enzymes by hybrid-type heteromeric dimers of site-directed mutants.

Farnesyl diphosphate synthase is a homodimer of subunits having typically two aspartate-rich motifs with two sets of substrate binding sites for an allylic diphosphate and isopentenyl diphosphate per molecule of a homodimeric enzyme. To determine whether each subunit contains an independent active site or whether the active sites are created by intersubunit interaction, we constructed several expression plasmids that overproduce hybrid-type heterodimers of Bacillus stearothermophilus FPP synthases constituting different types of mutated monomers, which exhibit little catalytic activity as homodimers, by combining two tandem fps genes for the manipulated monomer subunit with a highly efficient promoter trc within an overexpression pTrc99A plasmid. A heterodimer of a combination of subunits of the wild type and of R98E, a mutant subunit which exhibits little enzymatic activity as a dimer form (R98E)(2), exhibited 78% of the activity of the wild-type homodimer enzyme, (WT)(2). Moreover, when a hybrid-type heterodimeric dimer of FPP synthase mutant subunits (R98E/F220A) was prepared, the FPP synthase activity was 18- and 390-fold of that of each of the almost inactive mutants as a dimeric enzymes, (R98E)(2) and (F220A)(2) [Koyama, T., et al. (1995) Biochem. Biophys. Res. Commun. 212, 681-686], respectively. These results suggest that the subunits of the FPP synthase interact with each other to form a shared active site in the homodimer structure rather than an independent active site in each subunit.

Alkyl and Aryl Transferases↗
PubMed2004

Inhibition of isoprene biosynthesis pathway enzymes by phosphonates, bisphosphonates, and diphosphates.

We have investigated the docking of a variety of inhibitors and substrates to the isoprene biosynthesis pathway enzymes farnesyl diphosphate synthase (FPPS), isopentenyl diphosphate/dimethylallyl diphosphate isomerase (IPPI) and deoxyxylulose-5-phosphate reductoisomerase (DXR) using the Lamarckian genetic alogorithm program, AutoDock. The docked ligand structures are predicted with a approximately 0.8 A rms deviation from the structures determined crystallographically. The errors found are a function of the number of atoms in the ligand (R = 0.91, p < 0.0001) and, to a lesser extent, on the resolution of the crystallographic structure (R = 0.70, p < 0.008). The structures of three isoprenoid diphosphates docked to the FPPS enzyme reveal strong electrostatic interactions with Mg(2+), lysine and arginine active site residues. Similar results are obtained with the docking of four IPPI inhibitors to the IPPI enzyme. The DXR substrate, deoxyxylulose-5-phosphate, is found to dock to Mn(2+)-NADPH-DXR in an almost identical manner as does the inhibitor fosimdomycin to Mn(2+)-DXR (ligand heavy atom rms deviation = 0.90 A) and is poised to interact with NADPH. Bisphosphonate inhibitors are found to bind to the allylic binding sites in both eukaryotic and prokaryotic FPPSs, in good accord with recent crystallographic results (a 0.4 A rms deviation from the X-ray structure with the E. coli enzyme). Overall, these results show for the first time that the geometries of a broad variety of phosphorus-containing inhibitors and substrates of isoprene biosynthesis pathway enzymes can be well predicted by using computational methods, which can be expected to facilitate the design of novel inhibitors of these enzymes.

Aldose-Ketose Isomerases↗
PubMed1999

5'-Phosphoramidates and 5'-diphosphates of 2'-O-allyl-beta-D-arabinofuranosyluracil, -cytosine, and -adenine: inhibition of ribonucleotide reductase.

Continuing our studies on ribonucleotide reductase (RNR) mechanism-based inhibitors, we have now prepared the diphosphates (DP) of 2'-O-allyl-1-beta-D-arabinofuranosyl-uracil and -cytosine and 2'-O-allyl-9-beta-D-arabinofuranosyl-adenine and evaluated their inhibitory activity against recombinant murine RNR. 2'-O-Allyl-araUDP proved to be inhibitory to RNR at an IC(50) of 100 microM, whereas 2'-O-allyl-araCDP was only marginally active (IC(50) 1 mM) and 2'-O-allyl-araADP was completely inactive. The susceptibility of the parent nucleosides to phosphorylation by thymidine kinase and 2'-deoxycytidine kinase was also investigated, and all nucleosides proved to be poor substrates for the above-cited kinases. Moreover, prodrugs of 2'-O-allyl-araU and -araC monophosphates, namely 2'-O-allyl-5'-(phenylethoxy-L-alanyl phosphate)-araU and -araC, were prepared and tested against tumor cell proliferation but proved to be inactive. A molecular modeling study has been conducted in order to explain our results. The data confirm that for both the natural and analogue nucleoside diphosphates, the principal determinant interaction with the active site of RNR is with the diphosphate group, which forms strong hydrogen bonds with Glu623, Thr624, Ser625, and Thr209. Our findings indicate that the poor phosphorylation may represent an explanation for the lack of marked in vitro cytostatic activity of the test compounds.

Animals↗
PubMed2002

Synthesis of (E)-4-hydroxydimethylallyl diphosphate. An intermediate in the methyl erythritol phosphate branch of the isoprenoid pathway.

The syntheses of (E)-1-hydroxy-2-methyl-2-buten-4-yl diphosphate ((E)-4-hydroxydimethylallyl diphosphate, HDMAPP), an intermediate in the methyl erythritol phosphate pathway, and (E)-[4-(2)H]HDMAPP were accomplished in two steps from (E)-4-chloro-2-methyl-2-butenal. The synthetic route is easily adaptable for the facile incorporation of tritium at C-4 of the diphosphate.

Journal Article↗
PubMed1996

Cuprate-Mediated Synthesis and Biological Evaluation of Cyclopropyl- and tert-Butylfarnesyl Diphosphate Analogs.

The novel farnesyl diphosphate (FPP) analog 3-cyclopropyl-3-desmethylfarnesyl diphosphate (3-cpFPP, 1) was designed as a potential mechanism-based inhibitor of the FPP-utilizing enzyme protein-farnesyl transferase (PFTase). The key step in the synthesis of 1 involved the stereoselective coupling of vinyl triflate 8 with a lower order cyclopropyl cyanocuprate to afford the desired cyclopropyl ester 13. The sterically encumbered analog 3-desmethyl-3-tert-butylfarnesyl diphosphate (3-tbFPP, 7) was synthesized via a similar route. The use of the more reactive higher order tert-butyl cyanocuprate led to lower yields of ester 11, the key intermediate in the synthesis of 7. Biological evaluation of 3-cpFPP demonstrates that it is not a time-dependent inhibitor of recombinant yeast PFTase. Instead, 3-cpFPP is an alternative substrate for this enzyme that exhibits a K(m) comparable to FPP and a k(cat) only 5-fold lower than the natural substrate. In contrast, 3-tbFPP is an exceptionally poor substrate for yeast PFTase and acts as an inhibitor of this enzyme.

Journal Article↗
PubMed1999

Heterologous expression in Saccharomyces cerevisiae of an Arabidopsis thaliana cDNA encoding mevalonate diphosphate decarboxylase.

Sequence comparison with the mevalonate diphosphate decarboxylase (MVD) amino acid sequence of Saccharomyces cerevisiae identified an EST clone corresponding to a cDNA that may encode Arabidopsis thaliana MVD (AtMVD1). This enzyme catalyses the synthesis of isopentenyl diphosphate, the building block of sterol and isoprenoid biosynthesis, and uses mevalonate diphosphate as a substrate. Sequencing of the full-length cDNA was performed. The predicted amino acid sequence presents about 55% identity with the yeast, human and rat MVDs. The sequence of the genomic region of A. thaliana MVD was also obtained and Southern blot analysis on genomic DNA showed that A. thaliana could have at least one homologous MVD gene. In order to allow heterologous expression in S. cerevisiae, the MVD open reading frame (ORF) was then cloned under the control of the yeast PMA1 strong promoter. When expressed in yeast, the A. thaliana cDNA complemented both the thermosensitive MN19-34 strain deficient in MVD, and the lethal phenotype of an ERG19 deleted strain. However, the wild-type sterol content was not fully restored suggesting that the A. thaliana MVD activity may not be optimal in yeast. A two-hybrid assay was also performed to evaluate homodimer formation of the A. thaliana MVD and heterodimer formation between the plant and yeast heterologous enzymes.

Amino Acid Sequence↗
PubMed2003

Biosynthesis of anthecotuloide, an irregular sesquiterpene lactone from Anthemis cotula L. (Asteraceae) via a non-farnesyl diphosphate route.

Retrobiosynthetic analysis of the allergenic sesquiterpene lactone, anthecotuloide, suggested that this natural product could be formed either by head to head condensation of geranyl diphosphate with dimethylallyl diphosphate, or from farnesyl diphosphate (FPP), the accepted regular sesquiterpene precursor via the rearrangement of a germacranolide precursor. Isotopic labelling of anthecotuloide has now been achieved by feeding [1-(13)C]-glucose, [U-13C6]-glucose and [6,6-(2)H2]-glucose to aseptically grown plantlets of Anthemis cotula(family Asteraceae). Analysis of labelling patterns and absolute 13C abundances using quantitative 13C NMR spectroscopy showed that the isoprene building blocks of this sesquiterpene are formed exclusively via the MEP terpene biosynthetic pathway. This was supported by results from an experiment using [U-13C6]-glucose. A deuterium labelling experiment using [6,6-(2)H2]-glucose supported the original proposal and showed that anthecotuloide is formed from a non FPP precursor. Isotope ratio mass spectrometry suggested that there were two pathways for sesquiterpene biosynthesis in A. cotula.

Anthemis↗
PubMed2006

Incorporation of deuterium-labelled analogs of isopentenyl diphosphate for the elucidation of the stereochemistry of rubber biosynthesis.

A series of six deuterium-labelled analogs of isopententyl diphosphate (IPP) was prepared to investigate the detailed stereochemical course of addition of C5 units during rubber biosynthesis in Hevea brasiliensis and Parthenium argentatum. These analogs were incorporated into the cis-polyisoprene chain by rubber transferase in rubber particles, and the stereochemistry was determined by 2H-NMR analysis of the polymer or of levulinic acid derivatives obtained from its ozonolytic degradation. Results indicate that rubber chain elongation occurs with loss of the pro-S hydrogen of IPP, addition of the allylic diphosphate to the si face of IPP and inversion of stereochemistry at the carbon bearing the diphosphate.

Alkenes↗