Identification of the GGPS1 genes encoding geranylgeranyl diphosphate synthases from mouse and human.
(1/176)
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. (+info)
The African swine fever virus prenyltransferase is an integral membrane trans-geranylgeranyl-diphosphate synthase.
(2/176)
In a previous study, it was shown that the protein encoded by the gene B318L of African swine fever virus (ASFV) is a trans-prenyltransferase that catalyzes in vitro the condensation of farnesyl diphosphate and isopentenyl diphosphate to synthesize geranylgeranyl diphosphate and longer chain prenyl diphosphates (Alejo, A., Yanez, R. J., Rodriguez, J. M., Vinuela, E., and Salas, M. L. (1997) J. Biol. Chem. 272, 9417-9423). To investigate the in vivo function of the viral enzyme, we have determined, in this work, its subcellular localization and activity in cell extracts. Two systems were used in these studies: cells infected with ASFV and cells infected with a recombinant pseudo-Sindbis virus carrying the complete B318L gene. In this latter system, the trans-prenyltransferase was found to colocalize with the endoplasmic reticulum marker protein-disulfide isomerase, whereas in cells infected with ASFV, the viral enzyme was present in cytoplasmic viral assembly sites, associated with precursor viral membranes derived from the endoplasmic reticulum. In addition, after subcellular fractionation, the viral enzyme partitioned into the membrane fraction. Extraction of membrane proteins with alkaline carbonate and Triton X-114 indicated that the ASFV enzyme behaved as an integral membrane protein. The membrane enzyme synthesized predominantly all-trans-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate. These results indicate that the viral B318L protein is a trans-geranylgeranyl-diphosphate synthase, being the only enzyme of this type that is known to have a membrane localization. (+info)
Molecular analysis of prenyl chain elongating enzymes.
(3/176)
Multiple alignments of primary structures of many kinds of prenyltransferases that participate in the most fundamental prenyl-chain backbone synthesizing process in isoprenoid biosynthesis showed seven conserved regions in the primary structures of (E)-prenyl diphosphate synthases. However, no information has been available about the structures of (Z)-prenyl diphosphate synthases until our recent isolation of the gene for the undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26. The amino acid sequence of the (Z)-prenyl diphosphate synthase is totally different from those of (E)-prenyl chain elongating enzymes. Protein data base searches for sequences similar to that of the undecaprenyl diphosphate synthase yielded many unknown proteins which have not yet been characterized. Two of the proteins have recently been identified as the undecaprenyl diphosphate synthase of Escherichia coli and the dehydrodolichyl diphosphate synthase of Saccharomyces cerevisiae, indicating that there are three highly conserved regions in the primary structure of (Z)-prenyl chain elongating enzymes. (+info)
Novel prenyltransferase gene encoding farnesylgeranyl diphosphate synthase from a hyperthermophilic archaeon, Aeropyrum pernix. Molecularevolution with alteration in product specificity.
(4/176)
Prenyltransferases catalyse sequential condensations of isopentenyl diphosphate with allylic diphosphates. Previously, we reported the presence of farnesylgeranyl diphosphate (FGPP) synthase activity synthesizing C25 isoprenyl diphosphate in Natronobacterium pharaonis which is a haloalkaliphilic archaeon having C20-C25 diether lipids in addition to C20-C20 diether lipids commonly occurring in archaea [Tachibana, A. (1994) FEBS Lett. 341, 291-294]. Recently, it was found that a newly isolated aerobic hyperthermophilic archaeon, Aeropyrum pernix, had only C25-C25 diether lipids, not the usual C20-containing lipids [Morii, H., Yagi, H., Akutsu, H., Nomura, N., Sako, Y. & Koga, Y. (1999) Biochim. Biophys. Acta 1436, 426-436]. In this report, we describe the isoloation from A. pernix of the novel prenyltransferase gene, fgs, encoding FGPP synthase. The protein encoded by fgs was expressed in Escherichia coli as a glutathione S-transferase fusion protein and produced FGPP as a final product. Phylogenetic analysis of fgs with other prenyltransferases revealed that the short-chain prenyltransferase family is divided into three subfamilies: bacterial subfamily I, eukaryotic subfamily II, and archaeal subfamily III. fgs is clearly contained within the archaeal geranylgeranyl diphosphate (GGPP) synthase group (subfamily III), suggesting that FGPP synthase evolved from an archaeal GGPP synthase with an alteration in product specificity. (+info)
Prenyltransferase inhibitors block superoxide production by pulmonary vascular smooth muscle.
(5/176)
We recently showed that the farnesyltransferase inhibitor FTI-277 blocks interleukin 1beta (IL-1beta)-induced nitric oxide production in pulmonary vascular smooth muscle cells (SMC), whereas the geranylgeranyltransferase inhibitor GGTI-298 enhances this effect. Here we show that IL-1beta and platelet-derived growth factor (PDGF) stimulate superoxide production by pulmonary vascular SMC and that this effect is blocked by both FTI-277 and GGTI-298, suggesting that farnesylated and geranylgeranylated proteins are required for superoxide production. We also show that FTI-277 and GGTI-298 block superoxide production stimulated by constitutively active mutant H-Ras. Furthermore, superoxide production by IL-1beta, PDGF factor, and constitutively activated Ras is blocked by diphenyleneiodonium, implicating NAD(P)H oxidase as the generating enzyme. Given the role of oxidant radicals in vascular reactivity and injury, the action of both FTI-277 and GGTI-298 in suppressing superoxide generation by an inflammatory cytokine as well as by a potent smooth muscle mitogen may be therapeutically useful. (+info)
Ras protein of the slime mold Physarum polycephalum is farnesylated in vitro.
(6/176)
Physarum Ppras1 protein was efficiently prenylated by prenyltransferases of spinach. Surprisingly in spite of the C-terminal sequence (CLLL) specific for geranylgeranylation the protein was preferentially farnesylated. Consequences of this observation are discussed. (+info)
Isolation and characterization of the TERE1 gene, a gene down-regulated in transitional cell carcinoma of the bladder.
(7/176)
We have identified a novel cDNA product designated transitional epithelial response gene (TERE1), which was localized to chromosome 1p36. The TERE1 transcript (1.5 and 3.5 kb) is present in most normal human tissues including urothelium, but was reduced or absent in the majority of muscle invasive TCC tumors (22 out of 29 cases). The open reading frame encodes a protein of 338 amino acids (MW 36.8 KD). This protein is 57% homologous to a Drosophila protein called heix. We have shown by Western blotting and immuno-histochemistry with a polyclonal antibody to a specific TERE1 peptide, reduced or absent staining in muscle invasive tumors. Transfection of a sense TERE1 construct resulted in an 80-90% inhibition of cellular proliferation in two TCC cell lines and a lack of aneuploidy in the TERE1-transduced J82 cell line. These data suggest a potential role for this gene product in the progression of bladder cancer. (+info)
Isolation and functional analysis of homogentisate phytyltransferase from Synechocystis sp. PCC 6803 and Arabidopsis.
(8/176)
Tocopherols, collectively known as vitamin E, are lipid-soluble antioxidants synthesized exclusively by photosynthetic organisms and are required components of mammalian diets. The committed step in tocopherol biosynthesis involves condensation of homogentisic acid and phytyl diphosphate (PDP) catalyzed by a membrane-bound homogentisate phytyltransferase (HPT). HPTs were identified from Synechocystis sp. PCC 6803 and Arabidopsis based on their sequence similarity to chlorophyll synthases, which utilize PDP in a similar prenylation reaction. HPTs from both organisms used homogentisic acid and PDP as their preferred substrates in vitro but only Synechocystis sp. PCC 6803 HPT was active with geranylgeranyl diphosphate as a substrate. Neither enzyme could utilize solanesyl diphosphate, the prenyl substrate for plastoquinone-9 synthesis. In addition, disruption of Synechocystis sp. PCC 6803 HPT function causes an absence of tocopherols without affecting plastoquinone-9 levels, indicating that separate polyprenyltransferases exist for tocopherol and plastoquinone synthesis in Synechocystis sp. PCC 6803. It is surprising that the absence of tocopherols in this mutant had no discernible effect on cell growth and photosynthesis. (+info)