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.

Complete Tetrasaccharide Repeat Unit Biosynthesis of the Immunomodulatory Bacteroides fragilis Capsular Polysaccharide A

“…We found that characterization of these types of compounds by ESI-MS required in some cases as much as 4 nmoles, and in previous work we found that 200 nmoles would be required for NMR analysis. 17, 18 While, our work is dependent on the original structural characterizations of CPSA, 8 it does provide a simple, sensitive, non-radiolabel based method for preparing these materials and analyzing the specific roles of proteins involved in their formation. The major challenge now is to enhance MS signal, as sensitivity of this technique clearly decreased as the oligosaccharide repeat unit grew larger.…”

Functional identification of a galactosyltransferase critical to Bacteroides fragilis Capsular Polysaccharide A biosynthesis

“…( 14 ) These analogues were derived from a chemically synthesized 4-nitroaniline or 2-amideaniline geranyl diphosphate (2AA-GPP 4a, NAGPP 4b ) that was then elongated by the enzyme Undecaprenyl Pyrophosphate Synthase (UPPS) in the presence of isopentenyl diphosphate (IPP, 5 ). ( 15, 18 ) The long chain variable length isoprenoid product ( 6a-b ) of these reactions were then cleaved with a commercially available Alkaline Phosphatase to provide monophosphate substrate for WcfS (2AA-B(n)P, 7a , 4NA-B(n)P, 7b ). Each of the isoprenoid analogues were substrates for WcfS and the transfer of AADGal-phosphate was observed in a straight-forward reverse phase HPLC assay.…”

Tuning the Production of Variable Length, Fluorescent Polyisoprenoids Using Surfactant-Controlled Enzymatic Synthesis

“…18–20 However, the enzyme does not readily form shorter isoprenoids. Transient and steady-state kinetic studies of Escherichia coli UppS (UppS Ec ) have shown that surfactants have a profound influence on BPP formation.…”