Publications

• A conserved C-terminal RXG motif in the NgBR subunit of cis-prenyltransferase is critical for prenyltransferase activity.

  Grabinska K.A., Edani B.H., Park E.J., Kraehling J.R., Sessa W.C.

  <i>cis</i>-Prenyltransferases (<i>cis</i>-PTs) constitute a large family of enzymes conserved during evolution and present in all domains of life. In eukaryotes and archaea, <i>cis</i>-PT is the first enzyme committed to the synthesis of dolichyl phosphate, an obligate lipid carrier in protein gly ... >> More

  <i>cis</i>-Prenyltransferases (<i>cis</i>-PTs) constitute a large family of enzymes conserved during evolution and present in all domains of life. In eukaryotes and archaea, <i>cis</i>-PT is the first enzyme committed to the synthesis of dolichyl phosphate, an obligate lipid carrier in protein glycosylation reactions. The homodimeric bacterial enzyme, undecaprenyl diphosphate synthase, generates 11 isoprene units and has been structurally and mechanistically characterized in great detail. Recently, we discovered that unlike undecaprenyl diphosphate synthase, mammalian <i>cis</i>-PT is a heteromer consisting of NgBR (Nus1) and hCIT (dehydrodolichol diphosphate synthase) subunits, and this composition has been confirmed in plants and fungal <i>cis</i>-PTs. Here, we establish the first purification system for heteromeric <i>cis</i>-PT and show that both NgBR and hCIT subunits function in catalysis and substrate binding. Finally, we identified a critical R<i>X</i>G sequence in the C-terminal tail of NgBR that is conserved and essential for enzyme activity across phyla. In summary, our findings show that eukaryotic <i>cis</i>-PT is composed of the NgBR and hCIT subunits. The strong conservation of the R<i>X</i>G motif among NgBR orthologs indicates that this subunit is critical for the synthesis of polyprenol diphosphates and cellular function. << Less

  J. Biol. Chem. 292:17351-17361(2017) [PubMed] [EuropePMC]

• Molecular cloning, expression, and purification of undecaprenyl diphosphate synthase. No sequence similarity between E- and Z-prenyl diphosphate synthases.

  Shimizu N., Koyama T., Ogura K.

  Cloning of the gene for undecaprenyl diphosphate synthase was successful, providing the first primary structure for any prenyltransferase that catalyzes Z-prenyl chain elongation. A genomic DNA library of Micrococcus luteus B-P 26 was constructed in Escherichia coli, and the recombinant clones wer ... >> More

  Cloning of the gene for undecaprenyl diphosphate synthase was successful, providing the first primary structure for any prenyltransferase that catalyzes Z-prenyl chain elongation. A genomic DNA library of Micrococcus luteus B-P 26 was constructed in Escherichia coli, and the recombinant clones were grown on nylon membranes. The membrane was incubated directly by floating it on a reaction mixture containing radiolabeled isopentenyl diphosphate, nonlabeled farnesyl diphosphate, and Mg2+. Only the clones harboring plasmids encoding prenyltransferases could take up the substrates to synthesize and accumulate radiolabeled products inside the cells in amounts large enough to be detectable by autoradiography. Four positive colonies were found among about 4,000 bacterial colonies of the genomic DNA library. Two of them carried the gene for undecaprenyl diphosphate synthase, which catalyzes the Z-prenyl chain elongation, and the others carried the (all-E)-hexaprenyl diphosphate synthase genes (hexs-a and hexs-b; Shimizu, N., Koyama, T., and Ogura, K. (1998) J. Bacteriol. 180, 1578-1581). The undecaprenyl diphosphate synthase, which had a predicted molecular mass of 28.9 kDa, was overproduced in E. coli cells by applying a soluble expression system, and it was purified to near homogeneity. The deduced primary structure of the Z-prenyl chain-elongating enzyme is totally different from those of E-prenyl chain-elongating enzymes, which have characteristic conserved regions, including aspartate-rich motifs. << Less

  J. Biol. Chem. 273:19476-19481(1998) [PubMed] [EuropePMC]

• Mechanism of product chain length determination and the role of a flexible loop in Escherichia coli undecaprenyl-pyrophosphate synthase catalysis.

  Ko T.-P., Chen Y.-K., Robinson H., Tsai P.-C., Gao Y.-G., Chen A.P.-C., Wang A.H.-J., Liang P.-H.

  The Escherichia coli undecaprayl-pyrophosphate synthase (UPPs) structure has been solved using the single wavelength anomalous diffraction method. The putative substrate-binding site is located near the end of the betaA-strand with Asp-26 playing a critical catalytic role. In both subunits, an elo ... >> More

  The Escherichia coli undecaprayl-pyrophosphate synthase (UPPs) structure has been solved using the single wavelength anomalous diffraction method. The putative substrate-binding site is located near the end of the betaA-strand with Asp-26 playing a critical catalytic role. In both subunits, an elongated hydrophobic tunnel is found, surrounded by four beta-strands (betaA-betaB-betaD-betaC) and two helices (alpha2 and alpha3) and lined at the bottom with large residues Ile-62, Leu-137, Val-105, and His-103. The product distributions formed by the use of the I62A, V105A, and H103A mutants are similar to those observed for wild-type UPPs. Catalysis by the L137A UPPs, on the other hand, results in predominantly the formation of the C(70) polymer rather than the C(55) polymer. Ala-69 and Ala-143 are located near the top of the tunnel. In contrast to the A143V reaction, the C(30) intermediate is formed to a greater extent and is longer lived in the process catalyzed by the A69L mutant. These findings suggest that the small side chain of Ala-69 is required for rapid elongation to the C(55) product, whereas the large hydrophobic side chain of Leu-137 is required to limit the elongation to the C(55) product. The roles of residues located on a flexible loop were investigated. The S71A, N74A, or R77A mutants displayed 25-200-fold decrease in k(cat) values. W75A showed an 8-fold increase of the FPP K(m) value, and 22-33-fold increases in the IPP K(m) values were observed for E81A and S71A. The loop may function to bridge the interaction of IPP with FPP, needed to initiate the condensation reaction and serve as a hinge to control the substrate binding and product release. << Less

  J. Biol. Chem. 276:47474-47482(2001) [PubMed] [EuropePMC]

• Nogo-B receptor is necessary for cellular dolichol biosynthesis and protein N-glycosylation.

  Harrison K.D., Park E.J., Gao N., Kuo A., Rush J.S., Waechter C.J., Lehrman M.A., Sessa W.C.

  Dolichol monophosphate (Dol-P) functions as an obligate glycosyl carrier lipid in protein glycosylation reactions. Dol-P is synthesized by the successive condensation of isopentenyl diphosphate (IPP), with farnesyl diphosphate catalysed by a cis-isoprenyltransferase (cis-IPTase) activity. Despite ... >> More

  Dolichol monophosphate (Dol-P) functions as an obligate glycosyl carrier lipid in protein glycosylation reactions. Dol-P is synthesized by the successive condensation of isopentenyl diphosphate (IPP), with farnesyl diphosphate catalysed by a cis-isoprenyltransferase (cis-IPTase) activity. Despite the recognition of cis-IPTase activity 40 years ago and the molecular cloning of the human cDNA encoding the mammalian enzyme, the molecular machinery responsible for regulating this activity remains incompletely understood. Here, we identify Nogo-B receptor (NgBR) as an essential component of the Dol-P biosynthetic machinery. Loss of NgBR results in a robust deficit in cis-IPTase activity and Dol-P production, leading to diminished levels of dolichol-linked oligosaccharides and a broad reduction in protein N-glycosylation. NgBR interacts with the previously identified cis-IPTase hCIT, enhances hCIT protein stability, and promotes Dol-P production. Identification of NgBR as a component of the cis-IPTase machinery yields insights into the regulation of dolichol biosynthesis. << Less

  EMBO J. 30:2490-2500(2011) [PubMed] [EuropePMC]

• Undecaprenyl pyrophosphate synthetase from Lactobacillus plantarum: a dimeric protein.

  Muth J.D., Allen C.M.

  a++Undecaprenyl pyrophosphate synthetase has been purified from Lactobacillus plantarum. It catalyzes the formation of a C55 polyprenyl pyrophosphate having isoprene residues with cis stereochemistry. The enzyme was shown to be an acidic protein (pI = 5.1), which can be partially purified by prepa ... >> More

  a++Undecaprenyl pyrophosphate synthetase has been purified from Lactobacillus plantarum. It catalyzes the formation of a C55 polyprenyl pyrophosphate having isoprene residues with cis stereochemistry. The enzyme was shown to be an acidic protein (pI = 5.1), which can be partially purified by preparative gel electrophoresis and Blue-agarose column chromatography. The Km's of the enzyme for its substrates t,t-farnesyl pyrophosphate and isopentenyl pyrophosphate were determined to be 0.13 and 1.92 microM, respectively. The molecular weight of the enzyme was estimated by molecular sieve chromatography and gradient centrifugation to be 56,000 +/-4000. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the protein was composed of a dimer of 30,000-Da subunits. The enzyme was inactivated by the arginine-specific reagents phenylglyoxal, butanedione and, cyclohexanedione, but this inactivation was not prevented by either of the substrates. << Less

  Arch. Biochem. Biophys. 230:49-60(1984) [PubMed] [EuropePMC]

• Crystal structure of cis-prenyl chain elongating enzyme, undecaprenyl diphosphate synthase.

  Fujihashi M., Zhang Y.-W., Higuchi Y., Li X.-Y., Koyama T., Miki K.

  Undecaprenyl diphosphate synthase (UPS) catalyzes the cis-prenyl chain elongation onto trans, trans-farnesyl diphosphate (FPP) to produce undecaprenyl diphosphate (UPP), which is indispensable for the biosynthesis of bacterial cell walls. We report here the crystal structure of UPS as the only thr ... >> More

  Undecaprenyl diphosphate synthase (UPS) catalyzes the cis-prenyl chain elongation onto trans, trans-farnesyl diphosphate (FPP) to produce undecaprenyl diphosphate (UPP), which is indispensable for the biosynthesis of bacterial cell walls. We report here the crystal structure of UPS as the only three-dimensional structure among cis-prenyl chain elongating enzymes. The structure is classified into a protein fold family and is completely different from the so-called "isoprenoid synthase fold" that is believed to be a common structure for the enzymes relating to isoprenoid biosynthesis. Conserved amino acid residues among cis-prenyl chain elongating enzymes are located around a large hydrophobic cleft in the UPS structure. A structural P-loop motif, which frequently appears in the various kinds of phosphate binding site, is found at the entrance of this cleft. The catalytic site is determined on the basis of these structural features, from which a possible reaction mechanism is proposed. << Less

  Proc. Natl. Acad. Sci. U.S.A. 98:4337-4342(2001) [PubMed] [EuropePMC]