Domain organization of the polymerizing mannosyltransferases involved in synthesis of the Escherichia coli O8 and O9a lipopolysaccharide O-antigens

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DOIResolve DOI: http://doi.org/10.1074/jbc.M112.412577
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TypeArticle
Journal titleJournal of Biological Chemistry
ISSN0021-9258
1083-351X
Volume287
Issue45
Pages3813538149; # of pages: 15
SubjectBacteria; Biosynthesis; Glycoconjugate; Glycosyltransferases; Lipopolysaccharide (LPS); E. coli; O-polysaccharide; Mannosyltransferase; Modular Enzyme; Polymerase
AbstractThe Escherichia coli O9a and O8 polymannose O-polysaccharides (O-PSs) serve as model systems for the biosynthesis of bacterial polysaccharides by ATP-binding cassette transporter-dependent pathways. Both O-PSs contain a conserved primer-adaptor domain at the reducing terminus and a serotype-specific repeat unit domain. The repeat unit domain is polymerized by the serotype-specific WbdA mannosyltransferase. In serotype O9a, WbdA is a bifunctional α-(1→2)-, α-(1→3)-mannosyltransferase, and its counterpart in serotype O8 is trifunctional (α-(1→2), α-(1→3), and β-(1→2)). Little is known about the detailed structures or mechanisms of action of the WbdA polymerases, and here we establish that they are multidomain enzymes. WbdA^O9a contains two separable and functionally active domains, whereas WbdA^O8 possesses three. In WbdC^O9a and WbdB^O9a, substitution of the first Glu of the EX7E motif had detrimental effects on the enzyme activity, whereas substitution of the second had no significant effect on activity in vivo. Mutation of the Glu residues in the EX7E motif of the N-terminal WbdA^O9a domain resulted in WbdA variants unable to synthesize O-PS. In contrast, mutation of the Glu residues in the motif of the C-terminal WbdA^O9a domain generated an enzyme capable of synthesizing an altered O-PS repeat unit consisting of only α-(1→2) linkages. In vitro assays with synthetic acceptors unequivocally confirmed that the N-terminal domain of WbdA^O9a possesses α-(1→2)-mannosyltransferase activity. Together, these studies form a framework for detailed structure-function studies on individual domains and a strategy applicable for dissection and analysis of other multidomain glycosyltransferases.
Publication date
LanguageEnglish
AffiliationHuman Health Therapeutics; National Research Council Canada
Peer reviewedYes
NPARC number21269087
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Record identifiera086cbd3-35a4-4fb8-ad2d-e2b05c1372cd
Record created2013-12-05
Record modified2016-05-09
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