title: Mass spectrometric characterization of a two-glycosyltransferase tandem reaction for assembly of tetrasaccharide repeating unit of Escherichia coli O77 O-antigen.
abstract: The wbaD gene and wbaC gene from Escherichia coli O77 O-antigen gene cluster encoding mannosyltransferases were functionally characterized in vitro. A synthetic acceptor P(1)-(11-phenoxyundecyl)-P(2)-(2-acetamido-2-deoxy-α-D-glucopyranosyl) diphosphate (GlcNAc-PP-PhU) was used as an acceptor and GDP-Man as a donor substrate; the activities of WbaD and WbaC were confirmed by detailed structural characterization of their lipooligosacharide enzyme products using high-sensitivity negative-ion electrospray ionization (ESI) collision-induced dissociation tandem mass spectrometry (CID) MS-MS. The extensive fragmentation unequivocally demonstrated that the Man(1-3)-GlcNAc linkage in WbaD catalyzed reaction product and two Man(1-2)-Man linkages in tandem WbaD/WbaC catalyzed reaction product are present, respectively. This study provided valuable information for the understanding of diversified glycosyltransferase (GT) functions and the two GTs characterized can serve as additional enzyme sources for possible pharmaceutical related applications.
authors.affiliation: TEDA Institute of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, Tianjin 300457, China.
authors.forename: Dawei
authors.initials: D
authors.lastname: Zhou
authors.affiliation: TEDA Institute of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, Tianjin 300457, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Chen
authors.affiliation: TEDA Institute of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, Tianjin 300457, China.
authors.forename: Lingling
authors.initials: L
authors.lastname: Xu
authors.affiliation: N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prosp., 47, 119991 Moscow, Russia.
authors.forename: Natalia
authors.initials: N
authors.lastname: Utkina
authors.affiliation: N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prosp., 47, 119991 Moscow, Russia.
authors.forename: Leonid
authors.initials: L
authors.lastname: Danilov
authors.affiliation: N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prosp., 47, 119991 Moscow, Russia.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Torgov
authors.affiliation: N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prosp., 47, 119991 Moscow, Russia.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Veselovsky
authors.affiliation: TEDA Institute of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, Tianjin 300457, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
authors.affiliation: TEDA Institute of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, Tianjin 300457, China. Electronic address: fenglu63@nankai.edu.cn.
authors.forename: Lu
authors.initials: L
authors.lastname: Feng
mesh_terms: D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics; D019081:O Antigens / Q000737:chemistry*; D011135:Polysaccharides, Bacterial / Q000737:chemistry*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D029968:Escherichia coli Proteins; D019081:O Antigens; D011135:Polysaccharides, Bacterial; D016695:Glycosyltransferases; D005700:Galactosyltransferases; C505073:WbbD protein, E coli
keywords: E. coli O77; Electrospray ionization multistage tandem mass spectrometry; Glycosyltransferases; O-antigen repeating unit; WbaC; WbaD