Expression, Purification, and Characterization of a New Glucosyltransferase Involved in the Third Step of O-antigen Repeating-unit Biosynthesis of Escherichia Coli O152

Overview

Journal Glycoconj J

Publisher Springer

Specialties Biochemistry
Endocrinology

Date 2020 Jan 25

PMID 31974821

Authors

Chenying Dong

Diange Li

Ru Wang

Jian Chu

Zhongying Gong

Dawei Zhou

Affiliations

Soon will be listed here.

Abstract

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 α-PP-O(CH)CH) 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). 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.

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