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• W2055025159 abstract "The O-antigen polymerase of gram-negative bacteria has been difficult to characterize. Herein we report the biochemical and functional characterization of the protein product (Wzy) of the gene annotated as the putative O-antigen polymerase, which is located in the O-antigen biosynthetic locus of Francisella tularensis. In silico analysis (homology searching, hydropathy plotting, and codon usage assessment) strongly suggested that Wzy is an O-antigen polymerase whose function is to catalyze the addition of newly synthesized O-antigen repeating units to a glycolipid consisting of lipid A, inner core polysaccharide, and one repeating unit of the O-polysaccharide (O-PS). To characterize the function of the Wzy protein, a non-polar deletion mutant of wzy was generated by allelic replacement, and the banding pattern of O-PS was observed by immunoblot analysis of whole-cell lysates obtained by SDS-PAGE and stained with an O-PS-specific monoclonal antibody. These immunoblot analyses showed that O-PS of the wzy mutant expresses only one repeating unit of O-antigen. Further biochemical characterization of the subcellular fractions of the wzy mutant demonstrated that (as is characteristic of O-antigen polymerase mutants) the low molecular weight O-antigen accumulates in the periplasm of the mutant. Site-directed mutagenesis based on protein homology and topology, which was carried out to locate a catalytic residue of the protein, showed that modification of specific residues (Gly(176), Asp(177), Gly(323), and Tyr(324)) leads to a loss of O-PS polymerization. Topology models indicate that these amino acids most likely lie in close proximity on the bacterial surface." @default.
• W2055025159 created "2016-06-24" @default.
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• W2055025159 date "2010-09-01" @default.
• W2055025159 modified "2023-09-26" @default.
• W2055025159 title "Characterization of the O-antigen Polymerase (Wzy) of Francisella tularensis" @default.
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• W2055025159 doi "https://doi.org/10.1074/jbc.m110.143859" @default.
• W2055025159 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2934651" @default.
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