PMID- 31974821 OWN - NLM STAT- MEDLINE DCOM- 20210202 LR - 20210202 IS - 1573-4986 (Electronic) IS - 0282-0080 (Linking) VI - 37 IP - 2 DP - 2020 Apr TI - Expression, purification, and characterization of a new Glucosyltransferase involved in the third step of O-antigen repeating-unit biosynthesis of Escherichia coli O152. PG - 139-149 LID - 10.1007/s10719-020-09907-1 [doi] AB - The O antigen is indispensable for the full function and virulence of pathogenic bacteria. During O-repeating unit (RU) biosynthesis, committed glycosyltransferases (GTs) transfer various sugars from an activated sugar donor to the appropriate lipid carrier sequentially. While the nucleotide sequence specific for O antigen of pathogenic bacteria is already known, the exact substrate specificity of most hypothetical GTs have yet be characterized. In the present paper, we report the biochemical characterization of one alpha-glucosyltransferase, WfgE, a member of GT family 4. This enzyme is implicated in the pentasaccharide RU biosynthetic pathway of E. coli O152 O antigen. A chemoenzymatically synthesized acceptor (GlcGlcNAc alpha-PP-O(CH(2))(10)CH(3)) was used to characterize the WfgE activity. The enzyme product was determined to have a 1,2-linkage using strategy based on collision-induced dissociation electrospray ionization ion trap multiple tandem MS (CID-ESI-IT-MS(n)). The lack of a DxD motif and its high activity without divalent metal ions suggests that WfgE belongs to the GT-B fold superfamily. The enzyme is specific for beta-glucose or galactose-terminating acceptor substrates, and in particular UDP-glucose but also UDP-galactose as donor substrates. Our results suggest that WfgE catalyses the addition of the third sugar residue of the E. coli O152 O-RU. The recombinant GST-WfgE was solubilized and further purified to homogeneity via GST affinity chromatography, paving the way for structure-function relationship studies. FAU - Dong, Chenying AU - Dong C AD - Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, TEDA College, Nankai University, Tianjin, People's Republic of China. AD - TEDA Institue of Biological Sciences and Biotechnology, Nankai University, #23 HongDa Street, TEDA, Tianjin, 300457, China. AD - Tianjin Research Center for Functional Genomics and Biochips, TEDA College, Nankai University, Tianjin, People's Republic of China. AD - Tianjin Key Laboratory of Microbial Functional Genomics, TEDA College, Nankai University, Tianjin, People's Republic of China. FAU - Li, Diange AU - Li D AD - Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, TEDA College, Nankai University, Tianjin, People's Republic of China. AD - TEDA Institue of Biological Sciences and Biotechnology, Nankai University, #23 HongDa Street, TEDA, Tianjin, 300457, China. AD - Tianjin Research Center for Functional Genomics and Biochips, TEDA College, Nankai University, Tianjin, People's Republic of China. AD - Tianjin Key Laboratory of Microbial Functional Genomics, TEDA College, Nankai University, Tianjin, People's Republic of China. FAU - Wang, Ru AU - Wang R AD - Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, TEDA College, Nankai University, Tianjin, People's Republic of China. AD - TEDA Institue of Biological Sciences and Biotechnology, Nankai University, #23 HongDa Street, TEDA, Tianjin, 300457, China. AD - Tianjin Research Center for Functional Genomics and Biochips, TEDA College, Nankai University, Tianjin, People's Republic of China. AD - Tianjin Key Laboratory of Microbial Functional Genomics, TEDA College, Nankai University, Tianjin, People's Republic of China. FAU - Chu, Jian AU - Chu J AD - School of Automation and Electrical Engineering, Tianjin University of Technology and Education, Tianjin, People's Republic of China. FAU - Gong, Zhongying AU - Gong Z AD - Department of Neurology, Tianjin First Center Hospital, Tianjin, People's Republic of China. 13212121250@163.com. FAU - Zhou, Dawei AU - Zhou D AUID- ORCID: 0000-0002-3533-2027 AD - Key Laboratory of Molecular Microbiology and Technology of the Ministry of Education, TEDA College, Nankai University, Tianjin, People's Republic of China. daweizhou@nankai.edu.cn. AD - TEDA Institue of Biological Sciences and Biotechnology, Nankai University, #23 HongDa Street, TEDA, Tianjin, 300457, China. daweizhou@nankai.edu.cn. AD - Tianjin Research Center for Functional Genomics and Biochips, TEDA College, Nankai University, Tianjin, People's Republic of China. daweizhou@nankai.edu.cn. AD - Tianjin Key Laboratory of Microbial Functional Genomics, TEDA College, Nankai University, Tianjin, People's Republic of China. daweizhou@nankai.edu.cn. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20200123 PL - United States TA - Glycoconj J JT - Glycoconjugate journal JID - 8603310 RN - 0 (Escherichia coli Proteins) RN - 0 (O Antigens) RN - 2956-16-3 (Uridine Diphosphate Galactose) RN - EC 2.4.1.- (Glucosyltransferases) RN - V50K1D7P4Y (Uridine Diphosphate Glucose) SB - IM MH - Catalytic Domain MH - Escherichia coli Proteins/chemistry/genetics/*metabolism MH - Glucosyltransferases/chemistry/genetics/*metabolism MH - O Antigens/*biosynthesis MH - Substrate Specificity MH - Uridine Diphosphate Galactose/metabolism MH - Uridine Diphosphate Glucose/metabolism OTO - NOTNLM OT - Escherichia coli O-antigen OT - Functional characterization OT - Glucosyltransferase OT - Glycosyltransferase EDAT- 2020/01/25 06:00 MHDA- 2021/02/03 06:00 CRDT- 2020/01/25 06:00 PHST- 2019/09/24 00:00 [received] PHST- 2020/01/14 00:00 [accepted] PHST- 2019/12/15 00:00 [revised] PHST- 2020/01/25 06:00 [pubmed] PHST- 2021/02/03 06:00 [medline] PHST- 2020/01/25 06:00 [entrez] AID - 10.1007/s10719-020-09907-1 [pii] AID - 10.1007/s10719-020-09907-1 [doi] PST - ppublish SO - Glycoconj J. 2020 Apr;37(2):139-149. doi: 10.1007/s10719-020-09907-1. Epub 2020 Jan 23.