In vitro bacterial polysaccharide biosynthesis: defining the functions of Wzy and Wzz.
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.
Woodward
Robert
R
Department of Chemistry, Ohio State University, Columbus, Ohio, USA.
Yi
Wen
W
Li
Lei
L
Zhao
Guohui
G
Eguchi
Hironobu
H
Sridhar
Perali Ramu
PR
Guo
Hongjie
H
Song
Jing Katherine
JK
Motari
Edwin
E
Cai
Li
L
Kelleher
Patrick
P
Liu
Xianwei
X
Han
Weiqing
W
Zhang
Wenpeng
W
Ding
Yan
Y
Li
Mei
M
Wang
Peng George
PG
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
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