Reconstitution of UDP-galactopyranose mutase with 1-deaza-FAD and 5-deaza-FAD: analysis and mechanistic implications.

The galactofuranose moiety found in many surface constituents of microorganisms is derived from UDP-D-galactopyranose (UDP-Galp) via a unique ring contraction reaction catalyzed by a FAD-dependent UDP-Galp mutase. When the enzyme is reduced by sodium dithionite, its catalytic efficiency increases significantly. Since the overall transformation exhibits no net change in the redox state of the parties involved, how the enzyme-bound FAD plays an active role in the reaction mechanism is puzzling. In this paper, we report our study of the catalytic properties of UDP-Galp mutase reconstituted with deaza-FADs. It was found that the mutase reconstituted with FAD or 1-deazaFAD has comparable activity, while that reconstituted with 5-deazaFAD is catalytically inactive. Because 5-deazaFAD is restricted to net two-electron process, yet FAD and 1-deazaFAD can undergo concerted two-electron as well as stepwise one-electron redox reactions, the above results support a radical mechanism for the mutase catalyzed reaction. In addition, the activity of the mutase reconstituted with FAD was found to increase considerably at high pHs. These observations have allowed us to propose a new mechanism involving one-electron transfer from the reduced FAD to an oxocarbenium intermediate generated by C-1 elimination of UDP to give a hexose radical and a flavin semiquinone. Subsequent radical recombination leads to a coenzyme-substrate adduct which may play a central role to facilitate the opening and recyclization of the galactose ring. A deprotonation step, accompanied or followed the electron transfer step, to increase the nucleophilicity of the flavin radical anion may account for the activity enhancement at pH > 8.