FIGURE 6.

HPLC chromatograms recorded during addition of tunicamycin to the PaMnaA-catalyzed (A) forward reaction (UDP-GlcNAc to UDP-ManNAc) and (B) reverse reaction (UDP-ManNAc to UDP-GlcNAc). Chromatograms with 0.5 µM (orange), 1 µM (yellow), 10 µM (light green), 50 µM (dark green), 100 µM (light blue) and 200 µM (dark blue) tunicamycin, in comparison to the blank (black) without addition of tunicamycin, are shown. UDP-ManNAc and UDP-GlcNAc can be clearly discerned based on their retention time at ∼3.3 min and ∼3.7 min, respectively. The shift to the left with increasing tunicamycin concentration is due to the presence of a gradually increasing amount of DMSO.