Development of a multicomponent kinetic assay of the early enzymes in the Campylobacter jejuni N-linked glycosylation pathway

作者：Morrison James P; Troutman Jerry M; Imperiali Barbara^*

来源：Bioorganic & Medicinal Chemistry, 2010, 18(23): 8167-8171.

DOI：10.1016/j.bmc.2010.10.020

• Multienzyme assay
• Protein glycosylation
• C. jejuni
• Bacillosamine

The human pathogen Campylobacter jejuni possesses a general N-linked glycosylation system that is known to play a role in pathogenicity; however, a detailed understanding of this role remains elusive. A considerable hindrance to studying bacterial N-glycosylation in vivo is the absence of small molecule inhibitors to reversibly control the process. This report describes a pathway-screening assay that targets the early enzymes of C. jejuni N-glycan biosynthesis that would enable identification of inhibitors to the first four steps in the pathway. The assay includes PglF, PglE, PglD, PglC, and PglA; the enzymes involved in the biosynthesis of an undecaprenyl diphosphate-linked disaccharide and monitors the transfer of [H-3] GalNAc from the hydrophilic UDP-linked carrier to the lipophilic UndPP-diNAcBac (2,4-diacetamido-2,4,6-trideoxyglucose). The optimized assay has a Z'-factor calculated to be 0.77, indicating a robust assay suitable for screening. The diacylglycerol kinase from Streptococcus mutans, which provides a convenient method for phosphorylating undecaprenol, has been included in a modified version of the assay thereby allowing the screen to be conducted with entirely commercially available substrates.

• 出版日期2010-12-1