Studies of UDP-galactopyranose mutase from Escherichia coli: An unusual role of reduced FAD in its catalysis

The galactofuranose moiety found in many surface constituents of microorgan isms is derived from UDP-D-galactopyranose (UDP-Galp) via a unique ring con traction reaction catalyzed by UDP-Galp mutase. This enzyme, which has been isolated from several bacterial sources, is a flavoprotein where the FAD c oenzyme is noncovalently bound. Since its catalysis:does not appear to invo lve a redox mechanism; whether the enzyme-bound FAD plays an active role in the reaction mechanism,has been obscure. To study this transformation, the corresponding E. coil mutase was purified, and the ring contraction produc t, UDP-Galf, was chemically synthesized. Using UDP-Galf as the substrate, a K-m of 194 mu M and a k(cat) of 1.5 s(-1) for the catalysis in the reverse direction were obtained. The preference of the reaction toward the pyranos e product was confirmed by an equilibrium constant of 0.057 in the forward direction; Interestingly, when the enzyme: was reduced by sodium dithionite , its catalytic efficiency was increased by more than 2 orders of magnitude . A comparable rate enhancement was also noted when the flavin coenzyme was selectively reduced by photoreduction in the presence of 5-deazariboflavin under anaerobic conditions. Since mutase with either oxidize or reduced FA D is active, the change of the redox state in FAD appears to affect only th e activity, but not the catalytic mechanism. It is conceivable that;eductio n of FAD may induce a favorable conformational change of the enzyme that ma y be more conducive to catalysis. It is also possible that the reduced flav in bears a higher electron density at N-1, which may then be used to stabil ize the transiently formed oxocarbenium ion intermediates to facilitate cat alysis. Whether structural effects, electronic effects, or a combination of both dictates the ability of FAD to enhance the rate of the mutase reactio n is an interesting, albeit challenging question. Nevertheless, the present Work has provided, for the first time, evidence indicating the active invo lvement of FAD in regulating the catalytic efficiency of this enzyme.