FIGURE 7.

Co-incubation of Pen and Pal with the UDP-GlcNAc (G) yields four molecular species as determined by time-resolved ¹H NMR. A, when co-incubation reaction was carried out up to 8 h, in addition to the keto and hydrated forms of UDP-4-keto-6-deoxy-l-AltNAc, another predominant hydrated form of UDP-4-keto-6-deoxy-d-GlcNAc was observed. The selected chemical shift region for the anomeric protons of reactant and product is shown between 5.75 and 5.35 ppm, and the diagnostic H-6″ peak of the dual enzyme final products “F” is shown between 1.70 and 1.20 ppm. The peaks annotated * belong to a signal of Pal's hydrated product (“H”) as an intermediate, and the peaks annotated ** belong to signal peaks of Pal's keto product (“K”). B, 2-h reaction of the single enzyme Pal incubated with UDP-6-deoxy-d-GlcNAc-5,6-ene, showing only UDP-4-keto-6-deoxy-l-AltNAc was produced but no UDP-4-keto-6-deoxy-d-GlcNAc. The peak labeled with # belongs to a contaminant.