UMP Release in the Reaction of PglC with UDP-diNAcBac.

Assays were performed with 4 µM PglC and 80 µM UDP-diNAcBac at room temperature for 20 min in assay buffer containing 50 mM Hepes at pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 0.1% Triton X-100, and 10% DMSO. PglC was varied from 2 to 10 µM for measuring the concentration dependence of UMP release. A similar assay with 1 µM PglC and 80 µM UDP-diNAcBac was carried out with quenching at shorter times (5, 15, 30, and 60 s) (SI Appendix, Fig. S2). Control assays were performed in the absence of PglC and UDP-diNAcBac, and also in the presence of 4 µM PglC and 80 µM of UDP-GlcNAc.