Molecular analysis of NAD+-dependent xylitol dehydrogenase from the zygomycetous fungus Rhizomucor pusillus and reversal of the coenzyme preference

Abstract

The zygomycetous fungus Rhizomucor pusillus NBRC 4578 is able to ferment not only D-glucose but also D-xylose into ethanol. Xylitol dehydrogenase from R. pusillus NBRC 4578 (RpXDH), which catalyzes the second step of D-xylose metabolism, was purified, and its enzymatic properties were characterized. The purified RpXDH preferred NAD+ as its coenzyme and showed substrate specificity for xylitol, D-sorbitol, and ribitol. cDNA cloning of xyl2 gene encoding RpXDH revealed that the gene included a coding sequence of 1,092 bp with a molecular mass of 39,185 kDa. Expression of the xyl2 in R. pusillus NBRC 4578 was induced by D-xylose, and the expression levels were increased with accumulation of xylitol. The xyl2 gene was expressed in Escherichia coli, and coenzyme preference of the recombinant RpXDH was reversed from NAD+ to NADP+ in the double mutant D205A/I206R by site-directed mutagenesis.