title: Rationally designed short polyisoprenol-linked PglB substrates for engineered polypeptide and protein N-glycosylation.
abstract: 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.
authors.affiliation: Department of Chemistry, Chemistry Research Laboratory, Oxford University , Oxford OX1 3TA United Kingdom.
authors.forename: Feng
authors.initials: F
authors.lastname: Liu
authors.forename: Balakumar
authors.initials: B
authors.lastname: Vijayakrishnan
authors.forename: Amirreza
authors.initials: A
authors.lastname: Faridmoayer
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Taylor
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Parsons
authors.forename: Gonçalo J L
authors.initials: GJ
authors.lastname: Bernardes
authors.forename: Michael
authors.initials: M
authors.lastname: Kowarik
authors.forename: Benjamin G
authors.initials: BG
authors.lastname: Davis
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D016123:Campylobacter jejuni / Q000201:enzymology*; D004286:Dolichols / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D006001:Glycoconjugates / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006602:Hexosyltransferases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004286:Dolichols; D006001:Glycoconjugates; D008565:Membrane Proteins; D010455:Peptides; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase