Identifcation of reaction center of glycosyl transferase through molecular evolutionary and solid NMR analysis

• Project/Area Number: 15310155
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Living organism molecular science
• Research Institution: National Institute of Genetics
• Principal Investigator: SAITOU Naruya National Institute of Genetics, Department of Population Genetics, Professor, 集団遺伝研究系, 教授 (30192587)
• Co-Investigator(Kenkyū-buntansha): TAKEKOSI Kiyonori University of Kyoto, Graduate School of Science, Associate Professor, 大学院・理学研究科, 助教授 (10206964)
• Project Period (FY): 2003 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥16,000,000 (Direct Cost: ¥16,000,000); Fiscal Year 2004: ¥3,800,000 (Direct Cost: ¥3,800,000); Fiscal Year 2003: ¥12,200,000 (Direct Cost: ¥12,200,000)
• Keywords: glycosvl transferase / solid NMR / molecular evolution / E.coli / 3D structure of protein

Research Abstract

We first conducted molecular evolutionary analyses of bacterial glycosyltransferase genes by using newly introduced PC cluster. We particularly focused on cluster of glycosyltransferase genes that define outer cell wall sugar structures that are know to vary from strain to strain of E. coli. Homology search was done by using these ca. 20 gycosyltransferase amino acid sequences as queries, and gene phylogenies were constructed for each homologous group. We found that one group of homologous genes changed their sugar specificities during evolution. This suggests that enzyme specificity was altered through a series of amino acid changes. We therefore chose waaO gene of E. coli K12 strain, and overexpressed its protein. The protein was purified with using His-tag affinity collum chromatography, and appropriate amount of purified protein was used for solid NMR analysis. Several clear native C13 peaks were observed using solid NMR method. We also added C13-labeled glucose to this purified protein, and found new C13 peak that may correspond to glucose-protein binding.

Research Products

• [Journal Article] 1H to 2H Uniform Cross-Polarization Nuclear magnetic Resonance Using 2H Lee-Goldburg Irradiation in Static Powders (2005)
  • Author(s): T.Mizuno
  • Journal Title: J. Chem. Phys. 122 Pages: 84322-84334
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] The Evolutionary History of Glycosyltransferase Genes. (2005)
  • Author(s): Kaneko M., Saitou N.
  • Journal Title: Comparative glycomics and life evolution. Glycoforum(Hirabayashi J. (ed.))
  • NAID: 10012193305
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Evolution of hominoids and the search for a genetic basis for creating humanness. (2005)
  • Author(s): Saitou N.
  • Journal Title: Cytogenetic and Genome Research vol.108 Pages: 16-21
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] 1H to 2H Uniform Cross-Polarization Nuclear Magnetic Resonance Using 2H Lee-Goldburg Irradiation in Static Powders. (2005)
  • Author(s): Mizuno T., Takegoshi K., Terao T.
  • Journal Title: J. Chem. Phys. 122 Pages: 84322-84334
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] 1H to 2H Uniform Cross-Polarization Nuclear Magnetic Resonance Using 2H Lee-Goldburg Irradiation in Static Powders (2005)
  • Author(s): T.Mizuno
  • Journal Title: J.Chem.Phys. 122 Pages: 84322-84334
• [Journal Article] Sequential Arrangement of γ-Valeolactone Enantiomers Enclathrated in Cholic Acids Channels as Studied by 13C Solid-State NMR : Elucidation of the Optical Resolution Mechanism (2004)
  • Author(s): S.Nakamura
  • Journal Title: J. Am. Chem. Socs. 126 Pages: 8766-8769
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Dynamic Nuclear Polarization by Electron Spins in the Photoexcited Triplet State : I. Attainment of Proton Polarization of 0.7 at 105 K in Naphthalene (2004)
  • Author(s): K.Takeda
  • Journal Title: J. Phys. Soc. Jpn. 73 Pages: 2313-2318
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Dynamic Nuclear Polarization by Electron Spins in the Photoexcited Triplet State : II. High Polarization of the Residual Protons in Deuterated Naphthale (2004)
  • Author(s): K.Takeda
  • Journal Title: J. Phys. Soc. Jpn. 73 Pages: 2319-2322
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Switching-angle Sample Spinning NMR Probe with a Commercially Available 20 kHz Spinning System (2004)
  • Author(s): T.Mizuno
  • Journal Title: J. Magn. Reson. 171 Pages: 15-19
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] 13C Nuclear Overhauser Polarization-Magic Angle Spinning (NOP-MAS) NMR Spectroscopy in uniformly 13C-labeled Solid Proteins (2004)
  • Author(s): E.Katoh
  • Journal Title: J. Am. Chem. Soc. 126 Pages: 3653-3657
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] DNA sequence and comparative analysis of chimpanzee chromosome 22 (2004)
  • Author(s): The International Chimpanzee Chromosome 22 Consortium [H.Watanabe, A.Fujiyama, M.Hattori, T.D.Taylor, A.Toyoda, Y.Kuroki, H.Noguchi, A.BenKahla, H.Lehrach, R.Sudbrak, M.Kube, S.Taenzer, P.Galgoczy, M.Platzer, M.Scharfe, G.Nordsiek, H.Blocker, I.Hellmann, P.Khaitovich, S.Paabo, R.Reinhardt, H, -J.Zheng, X.-L.Zhang, G.-F.Zhu, B.-F.Wang, G.Fu, S.-X.Ren9, G.-P.Zhao, Z.Chen, Y.-S.Lee, J.-E.Cheong, S.-H.Choi, K.-M.Wu, T.-T.Liu, K.-J.Hsiao, S.-F.Tsai, C.-G.Kim, S.Oota, T.Kitano, Y.Hohara, N.Saitou, H.-S.Park, S.-Y.Wang, M.-L.Yaspo, Y.Sakaki]
  • Journal Title: Nature vol.429 No.6990 Pages: 382-388
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Evolution of O alleles of the human ABO blood group gene. (2004)
  • Author(s): Roubinet F., Despiau S.-P., Calafell F., Jin F., Bertanpetit J., Saitou N., Blancher A.
  • Journal Title: Transfusion vol.44 no.5 Pages: 707-715
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Integrative annotation of 21,037 human genes validated by full-length cDNA clones. (2004)
  • Author(s): Imanishi T., many other authors including Saitou N., Sugano S.
  • Journal Title: PLoS Biology vol.2 issue 6 Pages: 1-20
  • NAID: 120004667172
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Human specific amino acid changes found in 103 protein coding genes. (2004)
  • Author(s): Kitano T., Liu Y.-H., Ueda S., Saitou N.
  • Journal Title: Molecular Biology, Evolution vol.21 no.5 Pages: 936-944
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] 13C Nuclear Overhauser Polarization-Magic Angle Spinning (NOP-MAS) NMR Spectroscopy in uniformly 13C-labeled Solid Proteins. (2004)
  • Author(s): Katoh E., Takegoshi K., Terao T.
  • Journal Title: J. Am. Chem. Soc. 126 Pages: 3653-3657
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Development of High-temperature and High-pressure Nuclear Magnetic Resonance Probe for Supercritical Water. (2004)
  • Author(s): Amita F., Takegoshi.K.
  • Journal Title: Rev. Sci. Instru. 75 Pages: 467-471
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Sequential Arrangement of γ-Valerolactone Enantiomers Enclathrated in Cholic Acids Channels as Studied by 13C Solid-State NMR: Elucidation of the Optical Resolution Mechanism. (2004)
  • Author(s): Nakamura S., Imashiro F., Takegoshi K., Terao T.
  • Journal Title: J. Am. Chem. Socs. 126 Pages: 8769-8766
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Dynamic Nuclear Polarization by Electron Spins in the Photoexcited Triplet State: I. Attainment of Proton Polarization of 0.7 at 105 K in Naphthalene. (2004)
  • Author(s): Takeda K., Takegoshi K., Terao T.
  • Journal Title: J. Phys. Soc. Jpn. 73 Pages: 2313-2318
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Dynamic Nuclear Polarization by Electron Spins in the Photoexcited Triplet State: II. High Polarization of the Residual Protons in Deuterated Naphthalene. (2004)
  • Author(s): Takeda K., Takegoshi K., Terao T.
  • Journal Title: J. Phys. Soc. Jpn. 73 Pages: 2319-2322
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Switching-angle Sample Spinning NMR Probe with a Commercially Available 20 kHz Spinning System. (2004)
  • Author(s): Mizuno T., Takegoshi K., Terao T.
  • Journal Title: J.Magn. Reson. 171 Pages: 15-19
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Sequential Arrangement of γ-Valerolactone Enantiomers Enclathrated in Cholic Acids Channels as Studied by 13C Solid-State NMR : Elucidation of the Optical Resolution Mechanism (2004)
  • Author(s): S.Nakamura
  • Journal Title: J.Am.Chem.Socs. 126 Pages: 8769-8766
• [Journal Article] Dynamic Nuclear Polarization by Electron Spins in the Photoexcited Triplet State : I. Attainment of Proton Polarization of 0.7 at 105 K in Naphthalene (2004)
  • Author(s): K.Takeda
  • Journal Title: J.Phys.Soc.Jpn. 73 Pages: 2313-2318
• [Journal Article] Dynamic Nuclear Polarization by Electron Spins in the Photoexcited Triplet State : II. High Polarization of the Residual Protons in Deuterated Naphthalene (2004)
  • Author(s): K.Takeda
  • Journal Title: J.Phys.Soc.Jpn. 73 Pages: 2319-2322
• [Journal Article] Switching-angle Sample Spinning NMR Probe with a Commercially Available 20kHz Spinning System (2004)
  • Author(s): T.Mizuno
  • Journal Title: J.Magn.Reson. 171 Pages: 15-19
• [Book] ゲノムと進化 (2004)
  • Author(s): 斎藤成也
  • Total Pages: 210
  • Publisher: 新曜社
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Amita F.: "Development of a High-temperature and High-pressure Nuclear Magnetic Resonance Probe for Supercritical Water"Rev.Sci.Instru.. 75. 467-471 (2004)
• [Publications] Katoh E.: "13C Nuclear Overhauser Polarization-Magic Angle Spinning (NOP-MAS) NMR Spectroscopy in uniformly 13C-labeled Solid Proteins"J.Am.Chem.Soc.. 126. 3653-3657 (2004)
• [Publications] Takegoshi K.: "13C-1HDipolar-Driven 13C-13C Recoupling without 13C rf lrradiation in NMR of Rotating Solids"J.Chem.Phys.. 118. 2325-2341 (2003)