title: Chemoenzymatic synthesis of an isoprenoid phosphate tool for the analysis of complex bacterial oligosaccharide biosynthesis.
abstract: Undecaprenyl Pyrophosphate Synthase (UPPS) is a key enzyme that catalyzes the production of bactoprenols, which act as membrane anchors for the assembly of complex bacterial oligosaccharides. One of the major hurdles in understanding the assembly of oligosaccharide assembly is a lack of chemical tools to study this process, since bactoprenols and the resulting isoprenoid-linked oligosaccharides lack handles or chromophores for use in pathway analysis. Here we describe the isolation of a new UPPS from the symbiotic microorganism Bacteroides fragilis, a key species in the human microbiome. The protein was purified to homogeneity and utilized to accept a chromophore containing farnesyl diphosphate analogue as a substrate. The analogue was utilized by the enzyme and resulted in a bactoprenyl diphosphate product with an easy to monitor tag associated with it. Furthermore, the diphosphate is shown to be readily converted to monophosphate using a common molecular biology reagent. This monophosphate product allowed for the investigation of complex oligosaccharide biosynthesis, and was used to probe the activity of glycosyltransferases involved in the well characterized Campylobacter jejuni N-linked protein glycosylation. Novel reagents similar to this will provide key tools for the study of uncharacterized oligosaccharide assemblies, and open the possibility for the development of rapid screening methodology for these biosynthetic systems.
authors.affiliation: Department of Chemistry, University of North Carolina at Charlotte, Charlotte, NC 28223, United States.
authors.forename: Donovan K
authors.initials: DK
authors.lastname: Lujan
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Stanziale
authors.forename: Anahita Z
authors.initials: AZ
authors.lastname: Mostafavi
authors.forename: Sunita
authors.initials: S
authors.lastname: Sharma
authors.forename: Jerry M
authors.initials: JM
authors.lastname: Troutman
mesh_terms: D000116:Acetylgalactosamine / Q000737:chemistry; D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001441:Bacteroides fragilis / Q000201:enzymology* / Q000378:metabolism; D055162:Biocatalysis; D016123:Campylobacter jejuni / Q000201:enzymology; D060326:Chemistry Techniques, Synthetic; D016695:Glycosyltransferases / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis*; D011106:Polyisoprenyl Phosphates / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D012717:Sesquiterpenes / Q000378:metabolism
chemical_list: D009844:Oligosaccharides; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004808:farnesyl pyrophosphate; D016695:Glycosyltransferases; D019883:Alkyl and Aryl Transferases; C023266:undecaprenyl pyrophosphate synthetase; D000116:Acetylgalactosamine