The capsular polysaccharide (CPS) structure of Campylobacter jejuni contributes to its robust fitness. Many strains contain heptose moieties in their CPS units. The precursor heptose is GDP-d-glycero-α-d-manno-heptose; modifications to the stereochemistry at C3-C6 as well as additions of methyl and phosphoramidate groups lend to the hypervariability of the C. jejuni CPS structures. Synthesis of GDP-d-glycero-α-d-manno-heptose has been described previously, but using enzymes from Aneurinibacillus thermoaerophilus DSM 10155. Here we describe the complete synthesis of GDP-d-glycero-α-d-manno-heptose using enzymes from C. jejuni NTCC 11168: Cj1152 and Cj1423-Cj1425. Our results yield kinetic parameters for these enzymes and outline a successful strategy for milligram-gram scale synthesis of GDP-d-glycero-α-d-manno-heptose. This achievement is critical for the characterization of other carbohydrate tailoring enzymes, which are expected to utilize GDP-d-glycero-α-d-manno-heptose for the biosynthesis of more complex carbohydrates in the CPS of C. jejuni.
biosynthesis, synthesis, structure, heptose, capsular polysaccharide, Campylobacter jejuni, modification, Enzymes, phosphoramidate, stereochemistry
NCBI PubMed ID: 31449400Publication DOI: 10.1021/acs.biochem.9b00548Journal NLM ID: 0370623Publisher: American Chemical Society
Correspondence: raushel@tamu.edu
Institutions: Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States
Methods: 13C NMR, 1H NMR, kinetics assays, 31P NMR, anion-exchange chromatography, MS, genetic methods, HPLC, UV, cloning, bioinformatic analysis, enzymatic synthesis
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