title: 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.
authors.affiliation: a Biotechnology Research Center , Toyama Prefectural University , Imizu , Japan.
authors.forename: Shino
authors.initials: S
authors.lastname: Yamasaki-Yashiki
authors.forename: Hidenobu
authors.initials: H
authors.lastname: Komeda
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Hoshino
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Asano
mesh_terms: D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D008954:Models, Biological; D015394:Molecular Structure; D009243:NAD / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics*; D020103:Rhizomucor / Q000737:chemistry / Q000201:enzymology*; D014994:Xylose / Q000737:chemistry
chemical_list: D003067:Coenzymes; D009243:NAD; D014994:Xylose; D010088:Oxidoreductases
keywords: Rhizomucor pusillus; coenzyme preference; d-xylose metabolism; xylitol dehydrogenase