title: Biochemical characterization of an α1,2-colitosyltransferase from Escherichia coli O55:H7.
abstract: Colitose, also known as 3,6-dideoxy-L-galactose or 3-deoxy-L-fucose, is one of only five naturally occurring 3,6-dideoxyhexoses. Colitose was found in lipopolysaccharide of a number of infectious bacteria, including Escherichia coli O55 & O111 and Vibrio cholera O22 & O139. To date, no colitosyltransferase (ColT) has been characterized, probably due to the inaccessibility of the sugar donor, GDP-colitose. In this study, starting with chemically prepared colitose, 94.6 mg of GDP-colitose was prepared via a facile and efficient one-pot two-enzyme system involving an L-fucokinase/GDP-L-Fuc pyrophosphorylase and an inorganic pyrophosphatase (EcPpA). WbgN, a putative ColT from E. coliO55:H5 was then cloned, overexpressed, purified and biochemically characterized by using GDP-colitose as a sugar donor. Activity assay and structural identification of the synthetic product clearly demonstrated that wbgN encodes an α1,2-ColT. Biophysical study showed that WbgN does not require metal ion, and is highly active at pH 7.5-9.0. In addition, acceptor specificity study indicated that WbgN exclusively recognizes lacto-N-biose (Galβ1,3-GlcNAc). Most interestingly, it was found that WbgN exhibits similar activity toward GDP-l-Fuc (kcat/Km= 9.2 min(-1)mM(-1)) as that toward GDP-colitose (kcat/Km= 12 min(-1)mM(-1)). Finally, taking advantage of this, type 1 H-antigen was successfully synthesized in preparative scale.
authors.affiliation: Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Wu
authors.affiliation: Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Guohui
authors.initials: G
authors.lastname: Zhao
authors.affiliation: Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Tiehai
authors.initials: T
authors.lastname: Li
authors.affiliation: Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Jingyao
authors.initials: J
authors.lastname: Qu
authors.affiliation: College of Life Science, Hebei Normal University, Shijiazhuang, Hebei 050024, China.
authors.forename: Wanyi
authors.initials: W
authors.lastname: Guan
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 30071, China.
authors.forename: Jiajia
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Cheng
authors.initials: C
authors.lastname: Ma
authors.affiliation: Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Xu
authors.initials: X
authors.lastname: Li
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 30071, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.affiliation: Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA 30303, USA State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Tianjin 30071, China lli22@gsu.edu pwang11@gsu.edu.
authors.forename: Peng G
authors.initials: PG
authors.lastname: Wang
authors.forename: Lei
authors.initials: L
authors.lastname: Li
mesh_terms: D003837:Deoxy Sugars / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006156:Guanosine Diphosphate Sugars / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D003837:Deoxy Sugars; D029968:Escherichia coli Proteins; D006156:Guanosine Diphosphate Sugars; C096550:guanosine diphosphate-colitose; C025141:colitose; D005964:Glucosyltransferases
keywords: Escherichia coli o55; GDP-colitose; H-antigen; colitosyltransferase; synthesis