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The lipid carrier specificity of the protein N-glycosylation enzyme C. jejuni PglB was tested using a logical, synthetic array of natural and unnatural C10, C20, C30, and C40 polyisoprenol sugar pyrophosphates, including those bearing repeating cis-prenyl units. Unusual, short, synthetically accessible C20 prenols (nerylnerol 1d and geranylnerol 1e) were shown to be effective lipid carriers for PglB sugar substrates. Kinetic analyses for PglB revealed clear K(M)-only modulation with lipid chain length, thereby implicating successful in vitro application at appropriate concentrations. This was confirmed by optimized, efficient in vitro synthesis allowing >90% of Asn-linked β-N-GlcNAc-ylated peptide and proteins. This reveals a simple, flexible biocatalytic method for glycoconjugate synthesis using PglB N-glycosylation machinery and varied chemically synthesized glycosylation donor precursors.

Original publication

DOI

10.1021/ja409409h

Type

Journal article

Journal

J Am Chem Soc

Publication Date

15/01/2014

Volume

136

Pages

566 - 569

Keywords

Bacterial Proteins, Biocatalysis, Campylobacter jejuni, Dolichols, Glycoconjugates, Glycosylation, Hexosyltransferases, Kinetics, Membrane Proteins, Models, Molecular, Peptides, Protein Engineering, Substrate Specificity