Isopentenyl Pyrophosphates Research Articles

A Novel Hydrolytic Activity of Tri-Functional Geranylgeranyl Pyrophosphate Synthase in Haematococcus pluvialis.

Under environmental stresses, Haematococcus pluvialis accumulates large amounts of carotenoids. Scale of carotenoid biosynthesis depends on availability of geranylgeranyl pyrophosphate (GGPP) precursor, which is supplied by GGPP synthase (GGPPS) through sequential 1'-4 condensation of three isopentenyl pyrophosphates (IPPs) into dimethylallyl pyrophosphate (DMAPP). Using IPP and DMAPP as substrates, a tri-functional HpGGPPS was identified in this study to promiscuously synthesize allylic prenyl pyrophosphates (PPPs), e.g. C10 geranyl pyrophosphate (GPP), C15 farnesyl pyrophosphate (FPP), and C20 GGPP. Intriguingly, HpGGPPS can utilize GPP or FPP as a single substrate to synthesize GGPP by hydrolyzing the allylic PPP substrate into C5 IPP. Transcription of HpGGPPS and key carotenogenesis genes, morphological transformation, and carotenoid biosynthesis were differentially induced by environmental stresses, while HpGGPPS's products were low in vivo, implying that most of PPP flux had been shunted into carotenoid biosynthesis. Hydrolyzing allylic PPP intermediates into C5 building blocks by promiscuous HpGGPPS may be a fail safe for carotenoid accumulation against environmental stress.

Modeling studies with Helicobacter pylori octaprenyl pyrophosphate synthase reveal the enzymatic mechanism of trans-prenyltransferases

Octaprenyl pyrophosphate synthase (OPPs), an enzyme belonging to the trans-prenyltransferases family, is involved in the synthesis of C40 octaprenyl pyrophosphate (OPP) by reacting farnesyl pyrophosphate (FPP) with five isopentenyl pyrophosphates (IPP). It has been reported that OPPs is essential for bacteria's normal growth and is a potential target for novel antibacterial drug design. Here we report the crystal structure of OPPs from Helicobacter pylori, determined by MAD method at 2.8Å resolution and refined to 2.0Å resolution. The substrate IPP was docked into HpOPPs structure and residues involved in IPP recognition were identified. The other substrate FPP, the intermediate GGPP and a nitrogen-containing bisphosphonate drug were also modeled into the structure. The resulting model shed some lights on the enzymatic mechanism, including (1) residues Arg87, Lys36 and Arg39 are essential for IPP binding; (2) residues Lys162, Lys224 and Gln197 are involved in FPP binding; (3) the second DDXXD motif may involve in FPP binding by Mg2+ mediated interactions; (4) Leu127 is probably involved in product chain length determination in HpOPPs and (5) the intermediate products such as GGPP need a rearrange to occupy the binding site of FPP and then IPP is reloaded. Our results also indicate that the nitrogen-containing bisphosphonate drugs are potential inhibitors of FPPs and other trans-prenyltransferases aiming at blocking the binding of FPP.

Crystal Structures of Undecaprenyl Pyrophosphate Synthase in Complex with Magnesium, Isopentenyl Pyrophosphate, and Farnesyl Thiopyrophosphate: ROLES OF THE METAL ION AND CONSERVED RESIDUES IN CATALYSIS

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the consecutive condensation reactions of a farnesyl pyrophosphate (FPP) with eight isopentenyl pyrophosphates (IPP), in which new cis-double bonds are formed, to generate undecaprenyl pyrophosphate that serves as a lipid carrier for peptidoglycan synthesis of bacterial cell wall. The structures of Escherichia coli UPPs were determined previously in an orthorhombic crystal form as an apoenzyme, in complex with Mg(2+)/sulfate/Triton, and with bound FPP. In a further search of its catalytic mechanism, the wild-type UPPs and the D26A mutant are crystallized in a new trigonal unit cell with Mg(2+)/IPP/farnesyl thiopyrophosphate (an FPP analogue) bound to the active site. In the wild-type enzyme, Mg(2+) is coordinated by the pyrophosphate of farnesyl thiopyrophosphate, the carboxylate of Asp(26), and three water molecules. In the mutant enzyme, it is bound to the pyrophosphate of IPP. The [Mg(2+)] dependence of the catalytic rate by UPPs shows that the activity is maximal at [Mg(2+)] = 1 mm but drops significantly when Mg(2+) ions are in excess (50 mm). Without Mg(2+), IPP binds to UPPs only at high concentration. Mutation of Asp(26) to other charged amino acids results in significant decrease of the UPPs activity. The role of Asp(26) is probably to assist the migration of Mg(2+) from IPP to FPP and thus initiate the condensation reaction by ionization of the pyrophosphate group from FPP. Other conserved residues, including His(43), Ser(71), Asn(74), and Arg(77), may serve as general acid/base and pyrophosphate carrier. Our results here improve the understanding of the UPPs enzyme reaction significantly.

Preliminary X-ray diffraction analysis of octaprenyl pyrophosphate synthase crystals from Thermotoga maritima and Escherichia coli.

Octaprenyl pyrophosphate synthase (OPPs) catalyzes the condensation of five isopentenyl pyrophosphates with farnesyl pyrophosphate to generate C(40) octaprenyl pyrophosphate. The enzymes from the hyperthermophilic bacterium Thermotoga maritima and from the mesophilic Escherichia coli were expressed in E. coli and the recombinant proteins were purified and crystallized. The T. maritima OPPs crystals belong to space group P42(1)2, with unit-cell parameters a = b = 151.53, c = 69.72 A. The E. coli OPPs crystals belong to space group C222(1), with unit-cell parameters a = 247.66, b = 266.10, c = 157.93 A. Diffraction data were collected at 100 K using synchrotron radiation and an in-house X-ray source. Structure determination of T. maritima OPPs has been carried out using MIR data sets at 2.8 A resolution. The asymmetric unit contains one dimer. An initial model with 280 residues per subunit has been built and refined to 2.28 A resolution. It shows mostly helical structure and resembles that of avian farnesyl pyrophosphate synthase.

Catalytic Mechanism Revealed by the Crystal Structure of Undecaprenyl Pyrophosphate Synthase in Complex with Sulfate, Magnesium, and Triton

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes chain elongation of farnesyl pyrophosphate (FPP) to undecaprenyl pyrophosphate (UPP) via condensation with eight isopentenyl pyrophosphates (IPP). UPPs from Escherichia coli is a dimer, and each subunit consists of 253 amino acid residues. The chain length of the product is modulated by a hydrophobic active site tunnel. In this paper, the crystal structure of E. coli UPPs was refined to 1.73 A resolution, which showed bound sulfate and magnesium ions as well as Triton X-100 molecules. The amino acid residues 72-82, which encompass an essential catalytic loop not seen in the previous apoenzyme structure (Ko, T.-P., Chen, Y. K., Robinson, H., Tsai, P. C., Gao, Y.-G., Chen, A. P.-C., Wang, A. H.-J., and Liang, P.-H. (2001) J. Biol. Chem. 276, 47474-47482), also became visible in one subunit. The sulfate ions suggest locations of the pyrophosphate groups of FPP and IPP in the active site. The Mg2+ is chelated by His-199 and Glu-213 from different subunits and possibly plays a structural rather than catalytic role. However, the metal ion is near the IPP-binding site, and double mutation of His-199 and Glu-213 to alanines showed a remarkable increase of Km value for IPP. Inside the tunnel, one Triton surrounds the top portion of the tunnel, and the other occupies the bottom part. These two Triton molecules may mimic the hydrocarbon moiety of the UPP product in the active site. Kinetic analysis indicated that a high concentration (>1%) of Triton inhibits the enzyme activity.

Facile and stereospecific synthesis of (S)- and (R)-[2-2H]isopentenyl pyrophosphates

(S)- and (R)-[2-2H]isopentenyl pyrophosphates, which are useful precursors for stereochemical studies on the biosynthesis of isoprenoids, were chemically synthesized in five steps via(S)- and (R)-3-methyl-2,3-epoxybutan-1-ols starting with 3,3-dimethylallyl alcohol, in 18% and 15% overall yields, respectively.

Partial purification and properties of prenyltransferase from Pisum sativum

Prenyltransferase (EC 2.5.1.1; assayed as farnesyl pyrophosphate synthetase)was purified 106-fold from an homogenate of 3-day-old seedlings of Pisum sativum. Some of the properties of the purified enzyme were determined and these differed in several significant respects from those reported for preparations from other sources, e.g. the apparent MW was 96000 ± 4000 and the preparation could be dissociated into two subunits of MW 45000 ± 3000. The total activity of the extractable enzyme went through a sharp maximum (in the range 1 to 28 days) 3 days after germination. Farnesyl pyrophosphate was formed in cell-free extracts of peas from either isopentenyl pyrophosphate alone, or this together with geranyl pyrophosphate (optimum yields 1.2 and 10% respectively). Use of [1- 14C]- and [4- 14C]-isopentenyl pyrophosphates as the sole substrates and degradation of the products showed that the crude extracts contained a pool of the biogenetic equivalent of 3,3-dimethylallyl pyrophosphate. No analogous pool of geranyl pyrophosphate could be detected.

Prenyltransferase from Saccharomyces cerevisiae. Purification to homogeneity and molecular properties.

Prenyltransferase (EC 2.5.1.1) has been purified to homogeneity from the supernatant fraction of yeast by ammonium sulfate fractionation, diethylaminoethyl-cellulose and hydroxylapatite chromatography, and column isoelectric focusing techniques. The active enzyme from isoelectric focusing columns emerged as a single symmetrical peak with specific activities 15- to 35-fold higher than previously reported preparations. The enzyme was found to be homogeneous by continuous polyacrylamide gel electrophoresis at pH 8.4 and discontinuous polyacrylamide gel electrophoresis at pH 6.9 as well as sodium dodecyl sulfate polyacrylamide electrophoresis at pH 7.0. By means of gel chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was shown to be a dimer with a molecular weight of 84,000 plus or minus 10%. The isoelectric point of the enzyme was determined to be 5.3. The enzyme synthesizes farnesyl and geranylgeranyl pyrophosphates from dimethylallyl, geranyl, and farnesyl pyrophosphates. Michaelis constants for the enzyme were 4, 8, and 14 mu M for isopentenyl, dimethylallyl, and geranyl pyrophosphates, respectively.

Biosynthesis of phytoene from isopentenyl and farnesyl pyrophosphates by a partially purified tomato enzyme system

The solubilization, stabilization, and partial purification of an enzyme system that synthesizes phytoene from isopentenyl pyrophosphate is reported. Solubilization and partial purification of the enzyme system was achieved through the preparation of an acetone powder of tomato plastids, extraction of the powder with buffer, fractionation of this extract with ammonium sulfate, and passage of the enzyme through a column of Sephadex G-200. Stabilization was effected by storage in the presence of dithiothreitol. Either isopentenyl pyrophosphate, or farnesyl pyrophosphate plus isopentenyl pyrophosphate, serve as substrate for phytoene synthesis. The presence of isopentenyl pyrophosphate is required for the incorporation of farnesyl pyrophosphate into phytoene. Incorporation of substrate into phytoene is maximum at pH 8.0 and 20 °, and the K m value for isopentenyl pyrophosphate is 1.33 × 10 −6 m. Mn ++ and Mg ++ are required for phytoene synthesis, but pyridine nucleotides are not. Synthesis of phytoene is inhibited by -SH inhibitors and it is stimulated by dithiothreitol. The enzyme system also synthesizes acid-labile terpenyl pyrophosphates from isopentenyl pyrophosphate. Cleavage of these compounds with alkaline phosphatase yields a mixture of C 15 and C 20 terpenols. The enzyme system also effects the condensation of isopentenyl and farnesyl pyrophosphate. C 20 terpenols are liberated on alkaline phosphatase treatment of the products.

The Purification and Properties of Pig Liver Geranyl Pyrophosphate Synthetase

Abstract The purification and properties of geranyl pyrophosphate synthetase are reported. This enzyme is purified simultaneously with the enzyme for the synthesis of farnesyl pyrophosphate. It does not, however, have geranylgeranyl pyrophosphate synthetase activity. This enzyme, obtained as a single band of protein by starch gel electrophoresis, catalyzes the formation of both geranyl and farnesyl pyrophosphates from dimethylallyl pyrophosphate and 4-14C-isopentenyl pyrophosphate. These products have been identified by paper and gas-liquid chromatography. A pH range of 7.0 to 7.8 is optimum for the reaction. A metal ion is required and either Mg++ or Mn++ may be used, but Mn++ inhibits the reaction at concentrations greater than 4 x 10-3 m. Km values for isopentenyl and dimethylallyl pyrophosphates are 1.25 x 10-6 and 2.2 x 10-6 m, respectively. N-Ethylmaleimide and p-chloromercuribenzoate strongly inhibit the synthetase reaction, but iodoacetamide has only a slight inhibitory effect. The s20,w value for the synthetase is 4.7.

Properties of farnesyl pyrophosphate synthetase of pig liver

The isolation and partial purification of farnesyl pyrophosphate synthetase of pig liver are described. This enzyme does not have isopentenyl pyrophosphate isomerase, geranyl pyrophosphate synthetase, or geranyl geranyl pyrophosphate synthetase activities. Farnesyl pyrophosphate synthetase catalyzes the formation of farnesyl pyrophosphate through the condensation of geranyl and isopentenyl pyrophosphates. The product of this reaction has been characterized by paper and gas-liquid chromatography. A pH of 7.0 is optimum for the reaction, and K m values for geranyl and isopentenyl pyrophosphates are 4.0 × 10 −6 and 2.0 × 10 −6 M, respectively. An absolute metal requirement has been established for this reaction and Mn ++ is approximately 40 times more effective than Mg ++ at low ion concentrations. At pH 7.0, 2 × 10 −3 M iodoacetamide does not inhibit the reaction, but 1 × 10 −5 M p-hydroxy mercuribenzoate completely inactivates the enzyme.