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 mol 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 (Ki approximately 10 microM) 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 (R) configuration is preferred. Therefore xylitol dehydrogenase shares structural and functional properties with medium-chain sorbitol dehydrogenases.
Lunzer
R
R
Division of Biochemical Engineering, Institute of Food Technology, Universität für Bodenkultur (BOKU), Muthgasse 18, A-1190 Wien, Austria.
Mamnun
Y
Y
Haltrich
D
D
Kulbe
K D
KD
Nidetzky
B
B
D002384:Catalysis; D050542:D-Xylulose Reductase; D004789:Enzyme Activation; D004795:Enzyme Stability; D007700:Kinetics; D007064:L-Iditol 2-Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D015003:Yeasts / Q000201:enzymology*; D015032:Zinc / Q000378:metabolism*
D009243:NAD; D013401:Sugar Alcohol Dehydrogenases; D007064:L-Iditol 2-Dehydrogenase; D050542:D-Xylulose Reductase; D015032:Zinc