Purification, kinetic characterization and involvement of tryptophan residue at the NADPH binding site of xylose reductase from Neurospora crassa

Heterologous Expression, Purification, and Characterization of a Highly Active Xylose Reductase from Neurospora crassa

“…Its catalytic efficiency with respect to NADPH was 7-fold higher than that of the next closest enzyme (C. tenuis XR) (23) and 11-fold higher than that of any of the other XRs. The XR gene heterologously expressed and characterized here does not appear to encode an XR previously isolated and characterized from N. crassa NCIM 870 (29). Due to the lack of genetic and sequence information from that protein, it is not certain that they are different, but there are several differences between the two proteins.…”

“…The subunit weights and apparent native weights of the two enzymes are significantly dissimilar (Table 3) (29) , and the steady-state kinetic constants are different (the previously isolated XR has a fourfold-lower catalytic efficiency with respect to NADPH). Additionally, the previously isolated XR showed no activity with NADH, and the two enzymes differ in their pH optima and K m values for xylose (29). Although these differences could be due to alternative mRNA splicing, posttranslational modifications in N. crassa, or the fusion of the His 6 tag, the multiplicity of XRs is common in yeasts and filamentous fungi, and there are several other open reading frames in the N. crassa genome with sig- nificant sequence identity to XRs.…”

“…Such is the case with Neurospora crassa, which is known to convert plant biomass to ethanol (3,22,27), suggesting the presence of D-xylose-metabolizing enzymes (28) though no XR gene has been identified. Only one XR protein has been isolated, but no genetic or protein sequence was determined (29), thus limiting the study and eliminating the possibilities of recombinant expression and large-scale purification.…”

Preliminary Characterization of Xylose Reductase Partially Purified by Reversed Micelles from <i>Candida tropicalis</i> IEC5-ITV, an Indigenous Xylitol-Producing Strain

“…Enzyme analysis by SDS-PAGE (Figure 3) showed a band present in AP II which coincided in mobility with one present in CE, both with a molecular weight of 32.42 kD which is within the molecular weight range reported for other XRs [20][21][22]. XRs have been found to be present also as a dimer with subunits approximately 36 kD in molecular weight [20][21][22][23] so it is necessary in our case to perform electrophoresis under native conditions to determine whether the XR of Candida tropicalis IEC5-ITV is composed of one or more subunits.…”

“…XRs have been found to be present also as a dimer with subunits approximately 36 kD in molecular weight [20][21][22][23] so it is necessary in our case to perform electrophoresis under native conditions to determine whether the XR of Candida tropicalis IEC5-ITV is composed of one or more subunits. The presence of two additional bands, with molecular weights of 54.95 and 66.06 kD, was also noted but to a lesser extent, indicating that XR is not the only protein extracted under these conditions.…”