title: Domain interactions control complex formation and polymerase specificity in the biosynthesis of the Escherichia coli O9a antigen.
abstract: The Escherichia coli O9a O-polysaccharide (O-PS) is a prototype for bacterial glycan synthesis and export by an ATP-binding cassette transporter-dependent pathway. The O9a O-PS possesses a tetrasaccharide repeat unit comprising two α-(1→2)- and two α-(1→3)-linked mannose residues and is extended on a polyisoprenoid lipid carrier by the action of a polymerase (WbdA) containing two glycosyltransferase active sites. The N-terminal domain of WbdA possesses α-(1→2)-mannosyltransferase activity, and we demonstrate in this study that the C-terminal domain is an α-(1→3)-mannosyltransferase. Previous studies established that the size of the O9a polysaccharide is determined by the chain-terminating dual kinase/methyltransferase (WbdD) that is tethered to the membrane and recruits WbdA into an active enzyme complex by protein-protein interactions. Here, we used bacterial two-hybrid analysis to identify a surface-exposed α-helix in the C-terminal mannosyltransferase domain of WbdA as the site of interaction with WbdD. However, the C-terminal domain was unable to interact with WbdD in the absence of its N-terminal partner. Through deletion analysis, we demonstrated that the α-(1→2)-mannosyltransferase activity of the N-terminal domain is regulated by the activity of the C-terminal α-(1→3)-mannosyltransferase. In mutants where the C-terminal catalytic site was deleted but the WbdD-interaction site remained, the N-terminal mannosyltransferase became an unrestricted polymerase, creating a novel polymer comprising only α-(1→2)-linked mannose residues. The WbdD protein therefore orchestrates critical localization and coordination of activities involved in chain extension and termination. Complex domain interactions are needed to position the polymerase components appropriately for assembly into a functional complex located at the cytoplasmic membrane.
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1 and.
authors.forename: Sean D
authors.initials: SD
authors.lastname: Liston
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1 and.
authors.forename: Bradley R
authors.initials: BR
authors.lastname: Clarke
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1 and.
authors.forename: Laura K
authors.initials: LK
authors.lastname: Greenfield
authors.affiliation: the Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
authors.forename: Michele R
authors.initials: MR
authors.lastname: Richards
authors.affiliation: the Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
authors.forename: Todd L
authors.initials: TL
authors.lastname: Lowary
authors.affiliation: From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1 and cwhitfie@uoguelph.ca.
authors.forename: Chris
authors.initials: C
authors.lastname: Whitfield
mesh_terms: D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry* / Q000378:metabolism; D008358:Mannose / Q000737:chemistry / Q000378:metabolism; D008780:Methyltransferases / Q000737:chemistry* / Q000378:metabolism; D046912:Multiprotein Complexes / Q000096:biosynthesis / Q000737:chemistry; D019081:O Antigens / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; D019081:O Antigens; D011135:Polysaccharides, Bacterial; C000595910:WbdD protein, E coli; D008780:Methyltransferases; D016695:Glycosyltransferases; D008358:Mannose
keywords: Carbohydrate Biosynthesis; Enzyme Structure; Glycobiology; Glycosyltransferase; Lipopolysaccharide (LPS); Microbiology; Polysaccharide; Protein Complex