title: The C-glycosyltransferase IroB from pathogenic Escherichia coli: identification of residues required for efficient catalysis.
abstract: Escherichia coli C-glycosyltransferase IroB catalyzes the formation of a CC bond between enterobactin and the glucose moiety of UDP-glucose, resulting in the production of mono-, di- and tri-glucosylated enterobactin (MGE, DGE, TGE). To identify catalytic residues, we generated a homology model of IroB from aligned structures of two similar C-glycosyltransferases as templates. Superposition of our homology model onto the structure of a TDP-bound orthologue revealed residue W264 as a possible stabilizer of UDP-glucose. D304 in our model was located near the predicted site of the glucose moiety of UDP-glucose. A loop containing possible catalytic residues (H65, H66, E67) was found at the predicted enterobactin-binding site. We generated IroB variants at positions 65-67, 264, and 304 and investigated variant protein conformations and enzymatic activities. Variants were found to have Tm values similar to wild-type IroB. Fluorescence emission spectra of H65A/H66A, E67A, and D304N were superimposable with wild-type IroB. However, the emission spectrum of W264L was blue-shifted, suggesting solvent exposure of W264. While H65A/H66A retained activity (92% conversion of enterobactin, with MGE as a major product), all other IroB variants were impaired in their abilities to glucosylate enterobactin: E67A catalyzed partial (29%) conversion of enterobactin to MGE; W264L converted 55% of enterobactin to MGE; D304N was completely inactive. Activity-impaired variants were found to bind enterobactin with affinities within 2.5-fold of wild-type IroB. Given our outcomes, we propose that IroB W264 and D304 are required for binding and orienting UDP-glucose, while E67, possibly supported by H65/H66, participates in enterobactin/MGE/DGE deprotonation.
authors: Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke St. W., Montreal, Quebec H4B 1R6, Canada.
authors: Daniel
authors: D
authors: Foshag
authors: Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke St. W., Montreal, Quebec H4B 1R6, Canada.
authors: Cory
authors: C
authors: Campbell
authors: Department of Chemistry and Biochemistry, Concordia University, 7141 Sherbrooke St. W., Montreal, Quebec H4B 1R6, Canada; Groupe de Recherche Axé sur la Structure des Protéines (GRASP), Canada. Electronic address: peter.pawelek@concordia.ca.
authors: Peter D
authors: PD
authors: Pawelek
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D004758:Enterobactin / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017262:Siderophores / Q000737:chemistry* / Q000378:metabolism; D040681:Structural Homology, Protein; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000737:chemistry* / Q000378:metabolism; D056689:Uropathogenic Escherichia coli / Q000737:chemistry* / Q000201:enzymology
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D017262:Siderophores; D004758:Enterobactin; D001224:Aspartic Acid; D018698:Glutamic Acid; D014364:Tryptophan; D005964:Glucosyltransferases; C497513:IroB protein, E coli; D014532:Uridine Diphosphate Glucose
keywords: Enterobactin; Glycosyltransferase; Salmochelin; Siderophore; UDP-glucose