The C-glycosyltransferase IroB from pathogenic Escherichia coli: Identification of residues required for efficient catalysis

作者：Foshag Daniel; Campbell Cory; Pawelek Peter D^*

来源：Biochimica et Biophysica Acta-Proteins and Proteomics, 2014, 1844(9): 1619-1630.

DOI：10.1016/j.bbapap.2014.06.010

• Siderophore
• Enterobactin
• Glycosyltransferase
• Salmochelin
• UDP-glucose

Escherichia coli C-glycosyltransferase IroB catalyzes the formation of a C-C 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 T-m 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 0304 are required for binding and orienting UDP-glucose, while E67, possibly supported by H65/H66, participates in enterobactin/MGE/DGE deprotonation.

• 出版日期2014-9