计算机辅助木糖还原酶定点突变的生物信息学分析

木糖还原酶（xylose reductase，XR）是木糖代谢生成乙醇途径中一个重要的酶，目前利用纤维素生成酒精的关键问题之一：木糖代谢过程中XR和木糖醇脱氢酶（xylitol dehydrogenase，XDH）的氧化还原不平衡。本研究借助生物信息学手段（酶三维结构建模、酶和辅酶分子对接），充分分析数据库资源，找到了一些可能影响XR酶活性或辅酶依赖性的关键氨基酸。毕赤氏酵母XR与NADP之间有Lys21（K）、Val222（V）、Glu223（E）、Phe236（F）和Thr273（T）；毕赤氏酵母XR与NAD之间有Val222（V）、Glu223（E）、Phe236（F）、Glu237（E）和Thr273（T）；热带假丝酵母XR与NADP之间有Asn278（N）和Arg282（R）。对比两种辅酶与毕赤氏酵母XR形成氢键的氨基酸，如果使毕赤氏酵母XR只与辅酶NAD结合，则可以将Lys21替换成其它的氨基酸，因Lys21在所有XR序列中完全保守，需要进行氨基酸替代模拟计算预实验，在确保酶三维结构不变及NAD可以结合XR的前提条件下替代Lys21；如果使毕赤氏酵母XR只与辅酶NADP结合，则可以将Glu237（不完全保守）替换成其它的氨基酸。另外，还可以根据需要将这些形成氢键的氨基酸进行组合替代。要改变热带假丝酵母XR的NADP依赖性，可以替代Asn278（N）和/或Arg282（R）（不完全保守）。本研究为进一步酶的理性设计（提高活性及改变辅酶依赖性）并在分子水平上对木糖还原酶进行改造打下了基础。

Bioinformatic analysis of xylose reductase for site-directed mutagenesis based on computer

Xylose is one of the major components of hemicellulose, the second most abundant carbohydrate polymer in nature. xylose reductase (XR) is a important enzyme in the xylose metabolic pathway for producing ethanol. 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 XR, which is mainly NADPH dependent, and xylitol dehydrogenase, which is specific for NAD in the xylose metabolic pathway. Some possible key amino acids that effect the activity or preferring coenzyme of XR were found by bioinformatics methods such as homology modeling method, molecular docking in present study. They are Lys21(K), Val222(V), Glu223(E), Phe236(F) and Thr273(T) between XR of Pichia stipitis and NADP, Val222(V), Glu223(E), Phe236(F),Glu237(E)and Thr273(T) between XR of Pichia stipitis and NAD, Asn278(N) and Arg282(R) between XR of Candida tropicalis and NADP. Comparing Pichia stipitis XR amino acids that participate in the formation of hydrogen bonds with NADP and NAD, Lys21 can be substitute by other amino acids in order to change the coenzyme of XR of Pichia stipitis from NADP to NAD, but its substitution should be simulated based on computer under the condition of no change of XR three-dimensional structure and capable NAD binding since it is invariable in the multiple alignment results; Glu237 can be substitute by other amino acids in order to change the coenzyme of XR of Pichia stipitis from NAD to NADP. In addition, combinatorial substitutions of the amino acids that form hydrogen bonds with NADP or NAD can be carried out based on the research purpose. Asn278(N) and Arg282(R) can be substitute by other amino acids in order to change the coenzyme of XR of Candida tropicalis from NADP to NAD since they are not invariable. The present study will provide information for further rational design of XR to change the coenzyme preferring and increase activity of XR.