Zhang Y, Lee H
Department of Environmental Biology, University of Guelph, Ontario, Canada.
FEMS Microbiol Lett. 1997 Feb 15;147(2):227-32. doi: 10.1111/j.1574-6968.1997.tb10246.x.
Xylose reductase catalyzes the reduction of xylose to xylitol and is known to play a pivotal role in pentose metabolism in yeasts. We previously showed that a cystein residue may be involved in binding of the coenzyme NADPH to the Pichia stipitis xylose reductase through chemical modification studies. The question arose as to which of the three cysteine residues in this enzyme may be involved in coenzyme binding. We cloned the XYL1 gene encoding xylose reductase from P. stipitis into the phagemid pEMBL18(+) suitable for site-directed mutagenesis. Each of the three cysteine residues (Cys19, Cys27 and Cys130) was individually mutated to serine. All three Cys-->Ser variants remained functional, but with reduced catalytic activity. Sensitivity of the P. stipitis xylose reductase to thiol-specific reagents was attributed to both Cys27 and Cys130 residues as substitution of either residue with Ser resulted in a significant but incomplete loss of sensitivity to PCMBS. The apparent Km values of the Cys variants for NADPH, NADH and xylose did not differ from those of the wild-type enzyme isolated from yeast by more than 4-fold. Our results suggest that none of the Cys residues are directly involved in NADPH binding, although Cys130 may reside in or near the coenzyme binding region and might play a role in coenzyme specificity.
木糖还原酶催化木糖还原为木糖醇，已知其在酵母的戊糖代谢中起关键作用。我们之前通过化学修饰研究表明，一个半胱氨酸残基可能参与辅酶NADPH与树干毕赤酵母木糖还原酶的结合。问题在于该酶的三个半胱氨酸残基中哪一个可能参与辅酶结合。我们将编码树干毕赤酵母木糖还原酶的XYL1基因克隆到适合定点诱变的噬菌粒pEMBL18(+)中。三个半胱氨酸残基（Cys19、Cys27和Cys130）中的每一个都分别突变为丝氨酸。所有三个Cys→Ser变体仍具有功能，但催化活性降低。树干毕赤酵母木糖还原酶对硫醇特异性试剂的敏感性归因于Cys27和Cys130残基，因为将任何一个残基替换为Ser都会导致对PCMBS的敏感性显著但不完全丧失。Cys变体对NADPH、NADH和木糖的表观Km值与从酵母中分离的野生型酶相比，差异不超过4倍。我们的结果表明，虽然Cys130可能位于辅酶结合区域内或附近，并且可能在辅酶特异性中起作用，但没有一个半胱氨酸残基直接参与NADPH结合。
