Characterization of recombinant xylitol dehydrogenase from Galactocandida mastotermitis expressed in Escherichia coli.
The plasmid-encoded gene of xylitol dehydrogenase from the yeast Galactocandida mastotermitis was expressed in Escherichia coli at 25 degrees C. Recombinant enzyme was isolated in 70% yield using two steps of biomimetic affinity chromatography with the dye ligand Procion Red HE3B immobilized onto Sepharose 4B-CL. Similar to natural enzyme, recombinant xylitol dehydrogenase is a functional homotetramer with a stoichiometric content of catalytic zinc in each 37-kDa subunit. Though lacking bound Mg(2+) found in xylitol dehydrogenase isolated from yeast cell extracts, the recombinant enzyme is as active and stable as the native enzyme. Stereospecificity of enzymic hydrogen transfer from NADH has been determined by 1H-NMR and is 4-pro-R. A detailed steady-state kinetic analysis has been carried out for the enzymic reaction, polyol+NAD(+)<-->ketose+NADH+H(+), at pH 7.5 and 25 degrees C using xylitol and D-xylulose, the physiological polyol-ketose pair, as well as D-sorbitol and D-fructose. Primary deuterium kinetic isotope effects on steady-state kinetic parameters for oxidation of D-sorbitol and reduction of D-fructose have been measured at pH 7.5. Combined results of initial-rate analysis and isotope effect studies suggest that the enzyme utilizes a preferentially ordered kinetic mechanism in which NAD(+) binds before D-sorbitol and D-fructose is released before NADH. Dissociation of NADH appears to be the main rate-limiting step for D-sorbitol oxidation under substrate-saturated reaction conditions.
Nidetzky
Bernd
B
Institute of Biotechnology, Graz University of Technology, Petersgasse 12/I, A-8010, Graz, Austria. bernd.nidetzky@tugraz.at
Helmer
Heidemarie
H
Klimacek
Mario
M
Lunzer
Regina
R
Mayer
Gerhard
G
D002175:Candida / Q000201:enzymology*; D050542:D-Xylulose Reductase; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D013401:Sugar Alcohol Dehydrogenases; D050542:D-Xylulose Reductase