Development of a multicomponent kinetic assay of the early enzymes in the Campylobacter jejuni N-linked glycosylation pathway
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Imperiali14_Morrison_BMC_2010.pdf
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Author(s)
Imperiali, Barbara
Morrison, James P.
Troutman, Jerry
Alternative Title
Development of a multicomponent kinetic assay of the early enzymes in Campylobacter jejuni N-linked glycosylation pathway
Date Issued
October 2010
Journal
Bioorganic & Medicinal Chemistry
Publisher
Elsevier
Citation
Morrison, James P.; Troutman, Jerry M. and Imperiali, Barbara. “Development of a Multicomponent Kinetic Assay of the Early Enzymes in the Campylobacter Jejuni N-Linked Glycosylation Pathway.” Bioorganic & Medicinal Chemistry 18, no. 23 (December 2010): 8167–8171. © 2010 Elsevier Ltd
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Author's final manuscript
Abstract
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 [³H]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.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Massachusetts Institute of Technology. Department of Chemistry
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DOI of Published Version
https://doi.org/10.1016/j.bmc.2010.10.020
