title: O-antigen polymerase adopts a distributive mechanism for lipopolysaccharide biosynthesis.
abstract: Bacterial lipopolysaccharide (LPS) is an essential cell envelope component for gram-negative bacteria. As the most variable region of LPS, O antigens serve as important virulence determinants for many bacteria and represent a promising carbohydrate source for glycoconjugate vaccines. In the Wzy-dependent O-antigen biosynthetic pathway, the integral membrane protein Wzy was shown to be the sole enzyme responsible for polymerization of O-repeat unit. Its catalytic mechanism, however, remains elusive. Herein, Wzy was successfully overexpressed in Escherichia coli with an N-terminal His10-tag. Blue native polyacrylamide gel electrophoresis (BN-PAGE) revealed that the Wzy protein exists in its native confirmation as a dimer. Subsequently, we chemo-enzymatically synthesized the substrates of Wzy, the lipid-PP-linked repeat units. Together with an optimized O-antigen visualization method, we monitored the production of reaction intermediates at varying times. We present here our result as the first biochemical evidence that Wzy functions in a distributive manner.
authors.affiliation: Center for Diagnostics and Therapeutics and Department of Chemistry, Georgia State University, Atlanta, GA, 30303, USA.
authors.forename: Guohui
authors.initials: G
authors.lastname: Zhao
authors.forename: Baolin
authors.initials: B
authors.lastname: Wu
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.forename: Peng George
authors.initials: PG
authors.lastname: Wang
mesh_terms: D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D016695:Glycosyltransferases / Q000235:genetics* / Q000378:metabolism*; D006602:Hexosyltransferases / Q000235:genetics* / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D011993:Recombinant Fusion Proteins; D016695:Glycosyltransferases; D006602:Hexosyltransferases; C085652:O-antigen polymerase; C513753:Wzy polymerase, E coli