title: Rational design of an aryl-C-glycoside catalyst from a natural product O-glycosyltransferase.
abstract: Because the sugar moieties of natural products are primarily O-linked, the hydrolytic sensitivity of the glycosidic linkage limits their therapeutic application. One potential solution to this problem is to replace the labile O-glycosidic bond with an enzymatically and chemically stable C-glycosidic bond. In this study, computational analysis of the O-glycosyltransferase LanGT2 and the C-glycosyltransferase UrdGT2 was used to predict the changes necessary to switch the O-glycosylating enzyme to a C-glycosyltransferase. By screening rationally designed LanGT2 mutants a number of LanGT2 variants with C-glycosyltransferase activity were identified. One variant, having 10 amino acid substitutions, revealed the primary region that determines O- versus C-glycosylation. By modeling the active site of this mutant and probing the role of active site residues with alanine substitutions, this work also illuminates the mechanistic features of O- and C-glycosylation.
authors.affiliation: Department of Pharmaceutical Biology und Biotechnology, Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Härle
authors.forename: Stefan
authors.initials: S
authors.lastname: Günther
authors.forename: Benjamin
authors.initials: B
authors.lastname: Lauinger
authors.forename: Monika
authors.initials: M
authors.lastname: Weber
authors.forename: Bernd
authors.initials: B
authors.lastname: Kammerer
authors.forename: David L
authors.initials: DL
authors.lastname: Zechel
authors.forename: Andriy
authors.initials: A
authors.lastname: Luzhetskyy
authors.forename: Andreas
authors.initials: A
authors.lastname: Bechthold
mesh_terms: D000595:Amino Acid Sequence; D000617:Aminoglycosides / Q000096:biosynthesis; D055162:Biocatalysis; D001688:Biological Products / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006027:Glycosides / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D000617:Aminoglycosides; D001688:Biological Products; D006027:Glycosides; D011993:Recombinant Fusion Proteins; C064648:landomycin A; D016695:Glycosyltransferases