Structural and functional properties of a yeast xylitol dehydrogenase, a Zn2+-containing metalloenzyme similar to medium-chain sorbitol dehydrogenases

The NAD(+)-dependent xylitol dehydrogenase from the xylose-assimilating yeast Galactocandida mastotermitis has been purified in high yield (80 %) and characterized. Xylitol dehydrogenase is a heteronuclear multimetal protein that forms homotetramers and contains 1 mol of Zn2+ ions and 6 mot of Mg2+ ions per mol of 37.4 kDa protomer. Treatment with chelating agents such as EDTA results in the removal of the Zn2+ ions with a concomitant loss of enzyme activity. The Mg2+ ions are not essential for activity and are removed by chelation or extensive dialysis without affecting the stability of the enzyme. Results of initial velocity studies at steady state for D-sorbitol oxidation and D-fructose reduction together with the characteristic patterns of product inhibition point to a compulsorily ordered Theorell-Chance mechanism of xylitol dehydrogenase in which coenzyme binds first and leaves last. At pH 7.5, the binding of NADH (K-1 approximate to 10 mu M) is approx. 80-fold tighter than that of NAD(+). Polyhydroxyalcohols require at least five carbon atoms to be good substrates of xylitol dehydrogenase, and the C-2 (S), C-3 (R) and C-4 (A) configuration is preferred. Therefore xylitol dehydrogenase shares structural and functional properties with medium-chain sorbitol dehydrogenases.