木糖还原酶定点突变设计的生物信息学分析

Bioinformatic Analysis of Xylose Reductase for Site-Directed Mutagenesis

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.文中借助生物信息学手段(同源建模、酶和辅酶分子对接),通过分析数据库资源,找到了一些影响XR活性或辅酶依赖性的关键氨基酸.结果表明:树干毕赤氏酵母XR与烟酰胺腺嘌呤二核苷酸磷酸(NADP)之间形成氢键的氨基酸有Lys21、Val222、Glu223、Phe236和Thr273,与烟酰胺腺嘌呤二核苷酸(NAD)之间形成氢键的氨基酸有Val222、Glu223、Phe236、Glu237和Thr273;突变Lys21(完全保守)使树干毕赤氏酵母XR只与辅酶NAD结合,突变Glu237(不完全保守)使树干毕赤氏酵母XR只与辅酶NADP结合;热带假丝酵母XR与NADP之间形成氢键的氨基酸有Asn278和Arg282(两者都不完全保守),要改变其NADP依赖性,可以替代Asn278和/或Arg282.

One of the key problems affecting the ethanol production from cellulose is the unbalanced redox between xylose reductase （XR） and xylitol dehydrogenase （XDH） in the xylose metabolic process. In this paper, some key amino acids that affect the activity or coenzyme specificity of XR are identified based on the database sources by using the bioinformatic methods such as the homology modeling and the molecular docking. The results indicate that （1） amino acids Lys21, Va1222, Glu223, Phe236 and Thr273 in Pichia stipitis XR have hydrogen bonding with nicotinamide adenine dinucleotide phosphate （NADP）, while amino acids Va1222, Glu223, Phe236, Glu237 and Thr273 have hydrogen bonding with nicotinamide adenine dinucleotide （NAD） ; （2） the mutagenesis of Lys21 （conserved） is likely to result in the binding of Pichia stipitis XR with NAD only, while that of Glu237 （ not conserved） is likely to result in the binding with NADP only; （3） unconserved amino acids Asn278 and Arg282 in Candida tropicalis XR have hydrogen bonding with NADP; and （4） the mutagenesis of Ash278 or/and Arg282 is likely to result in the unbinding of Candida tropicalis XR from NADP.