Hiroshi Sagami

Polyprenyl Diphosphate Synthases

It is noteworthy that in spite of the similarity of the reactions catalyzed by these prenyltransferases, the modes of expression of catalytic function are surprisingly different, varying according to the chain length and stereochemistry of reaction products. These enzymes are summarized and classified into four groups, as shown in Figure 13. Short-chain prenyl diphosphates synthases such as FPP and GGPP synthases require no cofactor except divalent metal ions, Mg2+ or Mn2+, which are commonly required by all prenyl diphosphate synthases. Medium-chain prenyl diphosphate synthases, including the enzymes for the synthesis of all-E-HexPP and all-E-HepPP, are unusual because they each consist of two dissociable dissimilar protein components, neither of which has catalytic activity. The enzymes for the synthesis of long-chain all-E-prenyl diphosphates, including octaprenyl (C40), nonaprenyl-(C45), and decaprenyl (C50) diphosphates, require polyprenyl carrier proteins that remove polyprenyl products from the active sites of the enzymes to maintain efficient turnovers of catalysis. The enzymes responsible for Z-chain elongation include Z,E-nonaprenyl-(C45) and Z,E-undecaprenyl (C55) diphosphate synthases, which require a phospholipid. The classification of mammalian synthases seems to be fundamentally similar to that of bacterial synthases except that no medium-chain prenyl diphosphate synthases are included. The Z-prenyl diphosphate synthase in mammalian cells is dehydrodolichyl PP synthase, which catalyzes much longer chain elongations than do bacterial enzymes. Dehydrodolichyl PP synthase will be a major target of future studies in this field in view of its involvement in glycoprotein biosynthesis.

Evidence for Covalent Attachment of Diphytanylglyceryl Phosphate to the Cell-surface Glycoprotein of Halobacterium halobium

In a previous study, we demonstrated the occurrence of novel proteins modified with a diphytanylglyceryl group in thioether linkage in Halobacterium halobium (Sagami, H., Kikuchi, A., and Ogura, K. (1995) J. Biol. Chem. 270, 14851–14854). In this study, we further investigated protein isoprenoid modification in this halobacterium using several radioactive tracers such as [3H]geranylgeranyl diphosphate. One of the radioactive bands observed on SDS-polyacrylamide gel electrophoresis corresponded to a periodic acid-Schiff stain-positive protein (200 kDa). Radioactive and periodic acid-Schiff stain-positive peptides (28 kDa) were obtained by trypsin digestion of the labeled proteins. The radioactive materials released by acid treatment of the peptides showed a similar mobility to dolichyl (C55) phosphate on a normal-phase thin-layer plate. However, radioactive hydrolysates obtained by acid phosphatase treatment co-migrated not with dolichol (C55–65), but with diphytanylglycerol on both reverse- and normal-phase thin-layer plates. The mass spectrum of the hydrolysate was also coincident with that of diphytanylglycerol. The partial amino acid sequences of the 28-kDa peptides were found in a fragment (amino acids 731–816) obtainable by trypsin cleavage of the known cell-surface glycoprotein of this halobacterium. These results indicate that the cell-surface glycoprotein (200 kDa) is modified with diphytanylglyceryl phosphate.

Human Geranylgeranyl Diphosphate Synthase cDNA CLONING AND EXPRESSION

Geranylgeranyl diphosphate (GGPP) synthase (GGPPSase) catalyzes the synthesis of GGPP, which is an important molecule responsible for the C20-prenylated protein biosynthesis and for the regulation of a nuclear hormone receptor (LXR·RXR). The human GGPPSase cDNA encodes a protein of 300 amino acids which shows 16% sequence identity with the known human farnesyl diphosphate (FPP) synthase (FPPSase). The GGPPSase expressed inEscherichia coli catalyzes the GGPP formation (240 nmol/min/mg) from FPP and isopentenyl diphosphate. The human GGPPSase behaves as an oligomeric molecule with 280 kDa on a gel filtration column and cross-reacts with an antibody directed against bovine brain GGPPSase, which differs immunochemically from bovine brain FPPSase. Northern blot analysis indicates the presence of two forms of the mRNA.

A Partial Deficiency of Dehydrodolichol Reduction Is a Cause of Carbohydrate-deficient Glycoprotein Syndrome Type I

Carbohydrate-deficient glycoprotein (CDG) syndrome type I is a congenital disorder that involves the underglycosylation of N-glycosylated glycoproteins (Yamashita, K., Ideo, H., Ohkura, T., Fukushima, K., Yuasa, I., Ohno, K., and Takeshita, K. (1993) J. Biol. Chem 268, 5783-5789). In an effort to further elucidate the biochemical basis of CDG syndrome type I in our patients, we investigated the defect in the multi-step pathway for biosynthesis of lipid-linked oligosaccharides (LLO) by the metabolic labeling method using [3H]glucosamine, [3H]mannose, and [3H]mevalonate. The LLO levels in synchronized cultures of fibroblasts from these patients were severalfold lower than those in control fibroblasts in the S phase, and the oligosaccharides released from LLO showed the same structural composition, Glc1∼3·Man9·GlcNAc·GlcNAc, in the case of both the patients and controls. The amount of [3H]mannose incorporated into mannose 6-phosphate, mannose 1-phosphate, and GDP-mannose was greater in fibroblasts from these patients than in the control fibroblasts in the G1 period, although the ratios of these acidic mannose derivatives as indicated by the relative levels of radioactivity were the same for the two types of fibroblasts. Furthermore, upon metabolic labeling with [3H]mevalonate, the level of [3H]dehydrodolichol in fibroblasts from these patients increased in the S phase, and the levels of [3H]dolichol and [3H]dolichol-PP oligosaccharides concomitantly decreased, although the chain length distribution of the respective dolichols and dehydrodolichols was the same in the two types of fibroblasts. These results indicate that the conversion of dehydrodolichol to dolichol is partially defective in our patients and that the resulting loss of dolichol leads directly to underglycosylation.

Variable product specificity of solanesyl pyrophosphate synthetase

Abstract The distribution of polyprenyl pyrophosphates synthesized by the action of solanesyl pyrophosphate synthetase from Micrococcus luteus is dramatically changed depending on the Mg++ concentration. When the metal ion concentration is higher than 5 mM, octaprenyl and solanesyl (nonaprenyl) pyrophosphate are synthesized predominantly. On the other hand, when the metal ion level is lower than 0.5 mM, a variety of polyprenyl pyrophosphates ranging in carbon chain length from C15 to C40 are formed. Heptaprenyl pyrophosphate is the longest of the products formed at 0.1 mM of Mg++.

Dolichols of rubber plant, ginkgo and pine

Abstract Using a two-plate thin-layer chromatography method, we analyzed polyisoprenoid alcohols (dolichols and polyprenols) of the rubber plant Hevea brasiliensis (angiosperm), and of ginkgo Ginkgo biloba and pine Pinus sylvestris (gymnosperms). Special attention was paid to the occurrence of dolichol in various tissues of different plants. Dolichols were found to occur in all of the tissues examined except for flowers of the rubber plant. The chain length distributions of dolichols in seeds, young roots, young shoots, young leaves and old leaves of the rubber plant were C70-C95, C85-C105, C80-C105, C75-C105 and C65-C90, respectively. In the case of ginkgo, the chain length distributions of dolichols in seeds, embryos, young and old leaves were C70-C90, C70-C85, C70-C90 and C80-C95, respectively. Pine seeds were found to contain dolichols with the chain length distribution of C70-C90. Two kinds of polyprenol families were detected in leaves of the rubber plant and ginkgo. The longer chain polyprenol family was also detected in seeds of the rubber plant, in seeds and embryos of ginkgo and in seeds of pine. The chain length distributions of the polyprenols were not necessarily the same as those of dolichols occurring in the same tissues.

Studies on geranylgeranyl diphosphate synthase from rat liver: specific inhibition by 3-azageranylgeranyl diphosphate.

Geranylgeranyl diphosphate synthase from rat liver was separated from farnesyl diphosphate synthase, the most abundant and widely occurring prenyltransferase, by DEAE-Toyopearl column chromatography. The enzyme catalyzed the formation of E,E,E-geranylgeranyl diphosphate (V) from isopentenyl diphosphate (II) and dimethylallyl diphosphate (I), geranyl diphosphate (III), or farnesyl diphosphate (IV) with relative velocities of 0.09:0.15:1. 3-Azageranylgeranyl diphosphate (VII), designed as a transition-state analog for the geranylgeranyl diphosphate synthase reaction, was synthesized and found to act as a specific inhibitor for this synthase, but not for farnesyl diphosphate synthase. Diphosphate V and its Z,E,E-isomer (VI) also inhibited geranylgeranyl diphosphate synthase, but the effect was not as striking as that of the aza analog VII. Specific inhibition of geranylgeranyl diphosphate synthase by VII was also observed in experiments with 100,000g supernatants of rat brain and liver homogenates which contained isopentenyl diphosphate isomerase and prenyltransferases including farnesyl diphosphate synthase as well as geranylgeranyl diphosphate synthase. For farnesyl:protein transferase from rat brain, however, the aza compound did not show a stronger inhibitory effect than E,E,E-geranylgeranyl diphosphate.

The biosynthesis of dehydrodolichyl phosphates by rat liver microsomes

Using improved conditions with rat liver microsomes in the presence of 20% glycerol and 2% Triton X-100 at pH 8.5 it was shown that dehydrodolichyl diphosphate and dehydrodolichyl phosphate were synthesized from isopentenyl diphosphate and farnesyl diphosphate. Small amounts of geranylgeranyl diphosphate and geranylgeranyl phosphate were also formed. The carbon chain lengths of the dehydrodolichyl diphosphate and dehydrodolichyl phosphate were identical (C80-C85). A kinetic study showed that dehydrodolichyl diphosphate formed from farnesyl diphosphate and isopentenyl diphosphate was subsequently hydrolyzed to dehydrodolichyl phosphate. As the concentration of isopentenyl diphosphate was increased from 1 to 50 microM, the chain-length distribution of dehydrodolichyl products shifted from C75-C80 to C80-C85. Addition of MgCl2 into the assay mixture decreased product formation, but did not affect the chain-length distribution (C80-C85). The shift of the chain-length distribution to the same as that observed in naturally occurring dolichol derivatives (C90-C95) was observed when Triton X-100 was omitted from the assay mixture, although deletion of the detergent decreased the enzyme activity. These results, which provide insight into optimal conditions for enzymatic synthesis of the dolichol chain, are discussed in the context of the in vivo pathway for dolichol biosynthesis.

Distribution of polyprenols and dolichols in soybean plant

Abstract Using a convenient two-plate thin layer chromatography method, polyprenols and dolichols in soybean plant were analysed. These polyisoprenoid alcohols were found to occur in different quantities from tissue to tissue. The leaves contained ficaprenols (C 50 , C 55 , C 60 ), glycinoprenols (C 45 , C 50 , C 55 ) and dolichols (C 80 , C 85 ) with the major being ficaprenols, while the shoots, roots and seeds contained only dolichols. Analysis of the subcellular distribution of these compounds in the leaves demonstrated that ficaprenols, glycinoprenols and dolichols were mostly located in the chloroplast. When the chloroplast fraction was incubated with a combination of [1- 14 C]isopentenyl diphosphate and farnesyl diphosphate, radioactive polyprenols composed of C 45 , C 50 , C 55 and C 70 , C 75 were formed, suggesting that biosynthesis site for these polyprenols is localized in the chloroplast. The shorter chain ficaprenols and the longer chain dolichols were also found in the leaves of spinach, perilla, parsley and evergreen magnolia, which are dicotyledonous plants.

Decaprenyl pyrophosphate synthetase from mitochondria of pig liver

Decaprenyl pyrophosphate synthetase which catalyzes the synthesis of all-trans-decaprenyl pyrophosphate from isopentenyl pyrophosphate and either farnesyl pyrophosphate or geranylgeranyl pyrophosphate has been partially purified from mitochondria of pig liver. This enzyme lacks dimethylallyl-transferring and geranyl-transferring activities.