Comparison of Predicted Epimerases and Reductases of the Campylobacter jejuni D-altro- and L-gluco-Heptose Synthesis Pathways

Uniquely modified heptoses found in surface carbohydrates of bacterial pathogens are potential therapeutic targets against such pathogens. Our recent biochemical characterization of the GDP-6-deoxy-D-manno- and GDP-6-deoxy-D-altro-heptose biosynthesis pathways has provided the foundation for elucidation of the more complex L-gluco-heptose synthesis pathway of Campylobacter jejuni strain NCTC 11168. In this work we use GDP-4-keto, 6-deoxy-D-lyxo-heptose as a surrogate substrate to characterize three enzymes predicted to be involved in this pathway: WcaG(NCTC) (also known as Cj1427), MlghB (Cj1430), and MlghC (Cj1428). We compare them with homologues involved in D-altro-heptose production: WcaG(81176) (formerly WcaG), DdahB (Cjj1430), and DdahC (Cjj1427). We show that despite high levels of similarity, the enzymes have pathway-specific catalytic activities and substrate specificities. MlghB forms three products via C3 and C5 epimerization activities, whereas its DdahB homologue only had C3 epimerase activity along its cognate pathway. MlghC is specific for the double C3/C5 epimer generated by MlghB and produces L-gluco-heptose via stereospecific C4 reductase activity. In contrast, its homologue DdahC only uses the C3 epimer to yield D-altro-heptose via C4 reduction. Finally, we show that WcaG(NCTC) is not necessary for L-gluco-heptose synthesis and does not affect its production by MlghB and MlghC, in contrast to its homologue WcaG(81176), that has regulatory activity on D-altro-heptose synthesis. These studies expand our fundamental understanding of heptose modification, provide new glycobiology tools to synthesize novel heptose derivatives with biomedical applications, and provide a foundation for the structure function analysis of these enzymes.