PURIFICATION, CHARACTERIZATION AND STRUCTURE-ANALYSIS OF NADPH-DEPENDENT D-XYLOSE REDUCTASES FROM CANDIDA-TROPICALIS

NADPH-dependent D-xylose reductases (XRs) from Candida tropicalis IFO 0618 were purified and characterized. Mono Q HPLC revealed three XR isomers. The K-m values of XR1, XR2 and XR3 for D-xylose were 37, 30 and 34 mM, and for NADPH 14, 18 and 9 mu M, respectively. NADH did not act as a cofactor. The specificities of the three XRs for several aldoses were essentially the same. Gel filtration and cross-linking analysis showed that both XR1 and XR2 were dimers composed of identical subunits. The pi values of XR1 and XR2 were estimated to be 4.15 and 4.10, respectively. Comparison of the peptide maps of XR1 and XR2 showed that the molecular weights of 8 fragments of lysylendopeptidase-digested XR1 and XR2 were essentially the same as each other. The amino acid composition of each XR was also very similar. The molecular weights of XR1 and XR2 by mass spectra analysis were 36,497.91 and 36,539.68, respectively. The amino acid sequences of two XR1 peptide fragments (Nos. 4 and 5) were highly homologous with those of Pichia stipitis XR and mammalian aldose reductases.