Deoxysugars in glycopeptide antibiotics: Enzymatic synthesis of TDP-L-epivancosamine in chloroeremomycin biosynthesis

The 2,3,6-trideoxysugar L-epivancosamine is the terminal sugar added to the aglycone scaffold in chloroeremomycin. a member of the vancomycin family o f glycopeptide antibiotics. Five proteins from the chloroeremomycin biosynt hetic cluster, ORF14 and ORF23 to ORF26. have been expressed heterologously in Escherichia coil and purified to near homogeneity, and each has been ch aracterized for an enzymatic activity. These five enzymes reconstitute the complete biosynthesis of TDP-L-epivancosamine from TDP-4-keto-6-deoxy-D-glu cose. This process involves C-2 deoxygenation, C-3 amination and methylatio n, C-5 epimerization, and C-4 ketoreduction. Intermediates and the final pr oduct of this pathway have been identified by mass spectrometry and NMR. Th e pathway established here represents the complete in vitro reconstitution of an unusual sugar for an important class of antibiotics and sets the grou ndwork for future combinatorial biosynthesis for new bioactive compounds.