title: Structure of the polyisoprenyl-phosphate glycosyltransferase GtrB and insights into the mechanism of catalysis.
abstract: The attachment of a sugar to a hydrophobic polyisoprenyl carrier is the first step for all extracellular glycosylation processes. The enzymes that perform these reactions, polyisoprenyl-glycosyltransferases (PI-GTs) include dolichol phosphate mannose synthase (DPMS), which generates the mannose donor for glycosylation in the endoplasmic reticulum. Here we report the 3.0 Å resolution crystal structure of GtrB, a glucose-specific PI-GT from Synechocystis, showing a tetramer in which each protomer contributes two helices to a membrane-spanning bundle. The active site is 15 Å from the membrane, raising the question of how water-soluble and membrane-embedded substrates are brought into apposition for catalysis. A conserved juxtamembrane domain harbours disease mutations, which compromised activity in GtrB in vitro and in human DPM1 tested in zebrafish. We hypothesize a role of this domain in shielding the polyisoprenyl-phosphate for transport to the active site. Our results reveal the basis of PI-GT function, and provide a potential molecular explanation for DPM1-related disease.
authors.affiliation: Department of Physiology and Cellular Biophysics, Columbia University, New York, New York 10032, USA.
authors.forename: Chiara
authors.initials: C
authors.lastname: Ardiccioni
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA.
authors.forename: Oliver B
authors.initials: OB
authors.lastname: Clarke
authors.affiliation: Department of Physiology and Cellular Biophysics, Columbia University, New York, New York 10032, USA.
authors.forename: David
authors.initials: D
authors.lastname: Tomasek
authors.affiliation: Department of Pharmacology and Physiology, George Washington University, Washington, District of Columbia 20037, USA.|Department of Integrative Systems Biology, George Washington University, Washington, District of Columbia 20037, USA.
authors.forename: Habon A
authors.initials: HA
authors.lastname: Issa
authors.affiliation: Department of Pharmacology and Physiology, George Washington University, Washington, District of Columbia 20037, USA.|Department of Integrative Systems Biology, George Washington University, Washington, District of Columbia 20037, USA.
authors.forename: Desiree C
authors.initials: DC
authors.lastname: von Alpen
authors.affiliation: Department of Pharmacology and Physiology, George Washington University, Washington, District of Columbia 20037, USA.|Department of Integrative Systems Biology, George Washington University, Washington, District of Columbia 20037, USA.
authors.forename: Heather L
authors.initials: HL
authors.lastname: Pond
authors.affiliation: NE-CAT and Department of Chemistry and Chemical Biology, Cornell University, Argonne National Laboratory, Argonne, Illinois 60439, USA.
authors.forename: Surajit
authors.initials: S
authors.lastname: Banerjee
authors.affiliation: NE-CAT and Department of Chemistry and Chemical Biology, Cornell University, Argonne National Laboratory, Argonne, Illinois 60439, USA.
authors.forename: Kanagalaghatta R
authors.initials: KR
authors.lastname: Rajashankar
authors.affiliation: New York Structural Biology Center, X4 Beamlines, Brookhaven National Laboratory, Upton, New York 11973, USA.
authors.forename: Qun
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Ziqiang
authors.initials: Z
authors.lastname: Guan
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Chijun
authors.initials: C
authors.lastname: Li
authors.affiliation: New York Consortium on Membrane Protein Structure, New York Structural Biology Center, New York, New York 10027, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Kloss
authors.affiliation: New York Consortium on Membrane Protein Structure, New York Structural Biology Center, New York, New York 10027, USA.
authors.forename: Renato
authors.initials: R
authors.lastname: Bruni
authors.affiliation: Department of Informatics, Bioinformatics and Computational Biology, Garching 85748, Germany.|Institute for Advanced Study (TUM-IAS), TUM (Technische Universität München), Garching 85748, Germany.
authors.forename: Edda
authors.initials: E
authors.lastname: Kloppmann
authors.affiliation: Department of Informatics, Bioinformatics and Computational Biology, Garching 85748, Germany.|Institute for Advanced Study (TUM-IAS), TUM (Technische Universität München), Garching 85748, Germany.
authors.forename: Burkhard
authors.initials: B
authors.lastname: Rost
authors.affiliation: Department of Pharmacology and Physiology, George Washington University, Washington, District of Columbia 20037, USA.|Department of Integrative Systems Biology, George Washington University, Washington, District of Columbia 20037, USA.
authors.forename: M Chiara
authors.initials: MC
authors.lastname: Manzini
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032, USA.
authors.forename: Lawrence
authors.initials: L
authors.lastname: Shapiro
authors.affiliation: Department of Physiology and Cellular Biophysics, Columbia University, New York, New York 10032, USA.
authors.forename: Filippo
authors.initials: F
authors.lastname: Mancia
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D046939:Synechocystis / Q000201:enzymology*; D015027:Zebrafish
chemical_list: D016695:Glycosyltransferases; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase