, 12:665 | Cite as

A thermostable dolichol phosphoryl mannose synthase responsible for glycoconjugate synthesis of the hyperthermophilic archaeon Pyrococcus horikoshii

  • Yuji Urushibata
  • Shogo Ebisu
  • Ikuo Matsui
Original paper


Dolichol phosphoryl mannose synthase (DPM synthase) is an essential enzyme in the synthesis of N- and O-linked glycoproteins and the glycosylphosphatidyl-inositol anchor. An open reading frame, PH0051, from the hyperthermophilic archaeon Pyrococcus horikoshii encodes a DPM synthase ortholog, PH0051p. A full-length version of PH0051p was produced using an E. coli in vitro translation system and its thermostable activity was confirmed with a DPM synthesis assay, although the in vitro productivity was not sufficient for further characterization. Then, a yeast expression vector coding for the N-terminal catalytic domain of PH0051p was constructed. The N-terminal domain, named DPM(1-237), was successfully expressed, and turned out to be a membrane-bound form in Saccharomyces cerevisiae cells, even without its hydrophobic C-terminal domain. The membrane-bound DPM(1-237) was solubilized with a detergent and purified to homogeneity. The purified DPM(1-237) showed thermostability at up to 75°C and an optimum temperature of 60°C. The truncated mutant DPM(1-237) required Mg2+ and Mn2+ ions as cofactors the same as eukaryotic DPM synthases. By site-directed mutagenesis, Asp89 and Asp91 located at the most conserved motif, DXD, were confirmed as the catalytic residues, the latter probably bound to a cofactor, Mg2+. DPM(1-237) was able to utilize both acceptor lipids, dolichol phosphate and the prokaryotic carrier lipid C55-undecaprenyl phosphate, with Km values of 1.17 and 0.59 μM, respectively. The DPM synthase PH0051p seems to be a key component of the pathway supplying various lipid-linked phosphate sugars, since P. horikoshii could synthesize glycoproteins as well as the membrane-associated PH0051p in vivo.


Dolichol phosphoryl mannose synthase Membrane protein Hyperthermophilic archaea Pyrococcus Glycoprotein synthesis Glycoconjugate Glycosylphosphatidyl-inositol anchor Thermostable flippase 



This work was supported in part by the science program of the New Energy and Industrial Technology Development Organization, Japan. We thank Emiko Yamamoto for technical support.


  1. Abu-Qarn M, Eichler J (2006) Protein N-glycosulation in archaea: defining Haloferax volcani genes involved in S-layer glycoprotein glycosylation. Mol Microbiol 61:511–525PubMedCrossRefGoogle Scholar
  2. Ashida H, Maeda Y, Kinoshita T (2005) DPM1, the catalytic subunit of dolichol-phosphate mannose synthase, is tethered to and stabilized on the endoplasmic reticulum membrane by DPM3. J Biol Chem 281:896–904PubMedCrossRefGoogle Scholar
  3. Babczinski P, Haselbeck A, Tanner W (1980) Yeast mannosyl transferases requiring dolichyl phosphate and dolichyl phosphate mannose as substrate. Partial purification and characterization of the solubilized enzyme. Eur J Biochem 105:509–515PubMedCrossRefGoogle Scholar
  4. Burda P, Aebi M (1999) The dolichol pathway of N-linked glycosylation. Biocim Biophys Acta 1426:239–257Google Scholar
  5. Colussi PA, Taron CH, Mack J, Orlean P (1997) Human and Saccharomyces cerevisiae dolichol phosphate mannose synthases represent two classes of the enzymes, but both function in Schizosaccharomyces pombe. Proc Natl Acad Sci USA 94:7873–7878PubMedCrossRefGoogle Scholar
  6. Eichler J (2000) Novel glycoproteins of the halophilic archaeon Haloferax volcanii. Arch Microbiol 173:445–448PubMedCrossRefGoogle Scholar
  7. González JM, Masuchi Y, Robb FT, Ammerman JW, Maeder DL, Yanagibayashi M, Tamaoka J, Kato C (1998) Pyrococcus horikoshii sp. nov., a hyperthermophilic archaeon isolated from a hydrothermal vent at the Okinawa Trough. Extremophiles 2:123–130PubMedCrossRefGoogle Scholar
  8. Guan S, Bastin DA, Verma NK (1999) Functional analysis of the O antigen glucosylation gene cluster of Shigella flexneri bacteriophage SfX. Microbiology 145:1263–1273PubMedCrossRefGoogle Scholar
  9. Helenius J, Aebi M (2002) Transmembrane movement of dolichol linked carbohydrates during N-glycoprotein biosynthesis in the endoplasmic reticulum. Semin Cell Dev Biol 13:171–178PubMedCrossRefGoogle Scholar
  10. Higashibata H, Kikuchi H, Kawarabayashi Y, Matsui I (2003) Helicase and nuclease activities of hyperthermophile Pyrococcus horikoshii Dna2 inhibited by substrates with RNA segments at 5′-end. J Biol Chem 278:15983–15990PubMedCrossRefGoogle Scholar
  11. Ilgoutz SC, Zawadzki JL, Ralton JE, McConville MJ (1999) Evidence that free GPI glycolipids are essential for growth of Leishmania mexicana. EMBO J 18:2746–2755PubMedCrossRefGoogle Scholar
  12. Jensen JW, Schutzbach JS (1985) Activation of dolychyl-phospho-mannose synthase by phospholipids. Eur J Biochem 153:41–48PubMedCrossRefGoogle Scholar
  13. Kadowaki H, Kadowaki T, Wondisford FE, Taylor SI (1989) Use of polymerase chain reaction catalyzed by Taq DNA polymerase for site-specific mutagenesis. Gene 76:161–166PubMedCrossRefGoogle Scholar
  14. Kaiser C, Michaelis S, Mitcell A (1994) Lithium acetate yeast transformation. Methods in yeast genetics, a cold spring harbor laboratory course manual 1994 edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NYGoogle Scholar
  15. Kawarabayasi Y, Sawada M, Horikawa H, Haikawa Y, Hino Y, Yamamoto S, Sekine M, Baba S, Kosugi H, Hosoyama A, Nagai Y, Sakai M, Ogura K, Otsuka R, Nakazawa H, Takamiya M, Ohfuku Y, Funahashi T, Tanaka T, Kudoh Y, Yamazaki J, Kushida N, Oguchi A, Aoki K, Kikuchi H (1998) Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3. DNA Res 30:55–76CrossRefGoogle Scholar
  16. Koga Y, Morii H (2007) Biosynthesis of ether-type polar lipids in archaea and evolutionary considerations. Microbiol Mol Biol Rev 71:97–120PubMedCrossRefGoogle Scholar
  17. Korres H, Mavris M, Morona R, Manning PA, Verma NK (2005) Topological analysis of GtrA and GtrB proteins encoded by the serotype-converting cassette of Shigella flexneri. Biochem Biophys Res Commun 328:1252–1260PubMedCrossRefGoogle Scholar
  18. Kruszewska JS, Saloheimo M, Migdalski A, Orlean P, Penttila M, Palamarczyk G (2000) Dolichol phosphate mannose synthase from the filamentous fungus Trichoderma reesei belongs to the human and Schizosaccaromyces pombe class of the enzyme. Glycobiology 10:983–991PubMedCrossRefGoogle Scholar
  19. Maeda Y, Tanaka S, Hino J, Kangawa K, Kinoshita T (2000) Human dolichol-phosphate-mannose synthase consists of three subunits, DPM1, DPM2 and DPM3. EMBO J 19:2475–2482PubMedCrossRefGoogle Scholar
  20. Mazhari-Tabrizi R, Eckert V, Blank M, Muller R, Mumberg D, Funk M, Schwarz RT (1996) Cloning and functional expression of glycosyltransferases from parasitic protozoans by heterologous complementation in yeast: the dolichol phosphate mannose synthase from Trypanosoma brucei brucei. Biochem J 316:8583–8586Google Scholar
  21. Menon AK, Mayor S, Schwarz RT (1990) Biosynthesis of glycosyl-phosphatidylinositol lipids in Trypanosome brucei: involvement of mannosyl-phosphoryldolichol as the mannose donor. EMBO J 9:4249–4258PubMedGoogle Scholar
  22. Moens S, Vanderleyden J (1997) Glycoproteins in prokaryotes. Arch Microbiol 168:169–175PubMedCrossRefGoogle Scholar
  23. Orlean P (1990) Dolichol phosphate mannose synthase is required in vivo for glycosyl phosphatidylinositol membrane anchoring, O-mannosylation, and N-glycosylation of protein in Saccharomyces cerevisiae. Mol Cell Biol 10:5796–5805PubMedGoogle Scholar
  24. Orlean P, Albright C, Robbins PW (1988) Cloning and sequencing of the yeast gene for dolichol phosphate mannose synthase, an essential protein. J Biol Chem 263:17499–17507PubMedGoogle Scholar
  25. Perez M, Hirschberg CB (1986) Topography of glycosylation reactions in the rough endoplasmic reticulum membrane. J Biol Chem 261:6822–6830PubMedGoogle Scholar
  26. Prado-Figueroa M, Raper J, Opperdoes FR (1994) Possible localisation of dolichol-dependent mannosyltransferase of Trypanosoma brucei to the rough endoplasmic reticulum. Mol Biochem Parasitol 63:255–264PubMedCrossRefGoogle Scholar
  27. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NYGoogle Scholar
  28. Saxena IM, Brown RM Jr, Fevre M, Geremia RA, Henrissat B (1995) Multidomain architecture of beta-glycosyl transferases: implications for mechanism of action. J. Bacteriol 177:1419–1424PubMedGoogle Scholar
  29. Upreti RK, Kumar M, Shankar V (2003) Bacterial glycoproteins: functions, biosynthesis and applications. Proteomics 3:363–379PubMedCrossRefGoogle Scholar
  30. Villagómez-Castro JC, Calvo-Mendez C, Flores-Carreón A, López-Romero E (2000) Partial purification and characterization of dolichol phosphate mannose synthase from Entamoeba histolytica. Glycobiology 10:1311–1316PubMedCrossRefGoogle Scholar
  31. Weinberg WV, Schut G, Brehum S, Datta S, Adams MWW (2005) Cold shock of a hyperthermophilic archaeon: Pyrococcus furiosus exhibits multiple responses to a suboptimal growth temperature with a key role for membrane-bound glycoproteins. J. Bacteriol 187:336–348PubMedCrossRefGoogle Scholar
  32. Zhou GP, Troy FA (2005) NMR study of the preferred membrane orientation of polyisoplenols (dolichol) and the impact of their complex with polyisoprenyl recognition sequence peptides on membrane structure. Glycobiology 15:347–359PubMedCrossRefGoogle Scholar
  33. Zhu BCR, Laine RA (1996) Dolichyl-phosphomannose synthase from the archaeon Thermoplasma acidophilum. Glycobiology 6:811–816PubMedCrossRefGoogle Scholar
  34. Zimmerman JW, Robbins PW (1993) The hydrophobic domain of dolichyl-phosphate-mannose synthase is not essential for enzyme activity or growth in Saccharomyces cerevisiae. J Biol Chem 268:16746–16753PubMedGoogle Scholar

Copyright information

© Springer 2008

Authors and Affiliations

  1. 1.Biological Information Research CenterNational Institute of Advanced Industrial Science and TechnologyIbarakiJapan
  2. 2.ProteinExpress Co., Ltd.ChibaJapan

Personalised recommendations