title: In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz.
abstract: Polysaccharides constitute a major component of bacterial cell surfaces and play critical roles in bacteria-host interactions. The biosynthesis of such molecules, however, has mainly been characterized through in vivo genetic studies, thus precluding discernment of the details of this pathway. Accordingly, we present a chemical approach that enabled reconstitution of the E. coli O-polysaccharide biosynthetic pathway in vitro. Starting with chemically prepared undecaprenyl-diphospho-N-acetyl-D-galactosamine, the E. coli O86 oligosaccharide repeating unit was assembled by means of sequential enzymatic glycosylation. Successful expression of the putative polymerase Wzy using a chaperone coexpression system then allowed demonstration of polymerization in vitro using this substrate. Analysis of more substrates revealed a defined mode of recognition for Wzy toward the lipid moiety. Specific polysaccharide chain length modality was furthermore demonstrated to result from the action of Wzz. Collectively, polysaccharide biosynthesis was chemically reconstituted in vitro, providing a well defined system for further underpinning molecular details of this biosynthetic pathway.
authors.affiliation: Department of Chemistry, Ohio State University, Columbus, Ohio, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Woodward
authors.forename: Wen
authors.initials: W
authors.lastname: Yi
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.forename: Guohui
authors.initials: G
authors.lastname: Zhao
authors.forename: Hironobu
authors.initials: H
authors.lastname: Eguchi
authors.forename: Perali Ramu
authors.initials: PR
authors.lastname: Sridhar
authors.forename: Hongjie
authors.initials: H
authors.lastname: Guo
authors.forename: Jing Katherine
authors.initials: JK
authors.lastname: Song
authors.forename: Edwin
authors.initials: E
authors.lastname: Motari
authors.forename: Li
authors.initials: L
authors.lastname: Cai
authors.forename: Patrick
authors.initials: P
authors.lastname: Kelleher
authors.forename: Xianwei
authors.initials: X
authors.lastname: Liu
authors.forename: Weiqing
authors.initials: W
authors.lastname: Han
authors.forename: Wenpeng
authors.initials: W
authors.lastname: Zhang
authors.forename: Yan
authors.initials: Y
authors.lastname: Ding
authors.forename: Mei
authors.initials: M
authors.lastname: Li
authors.forename: Peng George
authors.initials: PG
authors.lastname: Wang
mesh_terms: D001428:Bacterial Vaccines / Q000138:chemical synthesis; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D016695:Glycosyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry; D010457:Peptidoglycan / Q000096:biosynthesis; D011134:Polysaccharides / Q000096:biosynthesis; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D013379:Substrate Specificity; D013682:Teichoic Acids / Q000096:biosynthesis / Q000737:chemistry
chemical_list: D001428:Bacterial Vaccines; D029968:Escherichia coli Proteins; D009844:Oligosaccharides; D010457:Peptidoglycan; D011134:Polysaccharides; D011135:Polysaccharides, Bacterial; D013682:Teichoic Acids; C489586:wzzB protein, E coli; C004275:colanic acid; D016695:Glycosyltransferases; C513753:Wzy polymerase, E coli