树干毕赤氏酵母木糖还原酶Lysine270突变的理性设计

木糖代谢过程中木糖还原酶（XR）和木糖醇脱氢酶（XDH）的氧化还原不平衡是利用纤维素生成酒精的关键问题之一.前期研究发现Lysine270是形成树干毕赤氏酵母木糖还原酶（P. stipitis XR）与NAD(P)结合口袋的关键氨基酸之一.为研究Lysine270对P. stipitis XR辅酶偏好性的影响,本研究将Lysine270用其它19种氨基酸替代,构建19种不同的XR突变子,利用同源建模和分子对接的方法评价不同突变子与NAD(P)之间的相互作用,并从中选择两个突变子K270R和K270N进行试验验证.树干毕赤氏酵母木糖还原酶K270R、K270N突变基因及野生基因WT-XYL1分别在大肠杆菌中进行了表达,表达后的蛋白经His-Tag纯化柱纯化后再进行辅酶偏好性及酶学性质研究.结果发现,Lysine270突变直接影响XR与NAD(P)的Km值,通过理性选择得到的K270N突变子的辅酶依赖性由NADPH完全逆转为NADH.

Mutational Study of the Role of Lysine 270 in the Pichia stipitis Xylose Reductas

Presently, one of the key problems of utilizing cellulose to produce ethanol is the impairment of the redox balance resulting from the different coenzyme specificities of xylose reductase (XR) and xylitol dehydrogenase in the xylose metabolic pathway. Lysine270 was found to be the key amino acid that formed the binding pocket of P. stipitis XR with NAD(P) in our previous study. To investigate the effect of Lysine270 on the XR coenzyme specificity, Lysine270 was substituted by the other 19 amino acids one by one, which would be producing 19 XR mutants. Further, the interaction between XR mutant and NAD(P) was estimated by homology modeling and molecular docking. Finally, K270R and K270N mutants were choosed to evaluate the bioinformatic analysis. The wild gene and mutagenesis genes were ligated into pET28b, and were transferred into E. coli BL21 (DE3). After being induced by IPTG, the xylose reductases were purified. Purified mutants K270R (Lys270→Arg), K270N (Lys270→Asn) were characterized by steady-state kinetic analysis. The results showed that the coenzyme dependence of K270N was completely reversed to NADH.