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 microorganisms is derived from UDP- d -galactopyranose (UDP-Gal p ) via a unique ring contraction reaction catalyzed by UDP-Gal p mutase. This enzyme, which has been isolated from several bacterial sources, is a flavoprotein where the FAD coenzyme is noncovalently bound. Since its catalysis does not appear to involve 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. coli mutase was purified, and the ring contraction product, UDP-Gal f, was chemically synthesized. Using UDP-Gal f as the substrate, a K m of 194 μM 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 pyranose 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 oxidized or reduced FAD is active, the change of the redox state in FAD appears to affect only the activity, but not the catalytic mechanism. It is conceivable that reduction of FAD may induce a favorable conformational change of the enzyme that may be more conducive to catalysis. It is also possible that the reduced flavin bears a higher electron density at N-1, which may then be used to stabilize the transiently formed oxocarbenium ion intermediates to facilitate catalysis. Whether structural effects, electronic effects, or a combination of both dictates the ability of FAD to enhance the rate of the mutase reaction is an interesting, albeit challenging question. Nevertheless, the present work has provided, for the first time, evidence indicating the active involvement of FAD in regulating the catalytic efficiency of this enzyme.