树干毕赤酵母木糖还原酶Lysine270定点突变的理性设计

Rational Design of Site-Directed Mutagenesis of Lysine270 in Pichia stipitis Xylose Reductase

Lysine270是树干毕赤酵母木糖还原酶(PsXR)与还原型烟酰胺腺嘌呤二核苷磷酸(NADPH)或还原型烟酰胺腺嘌呤二核苷酸(NADH)形成结合口袋的关键氨基酸之一.为研究该位点对PsXR辅酶偏好性的影响,用其它19种氨基酸替代Lysine270,构建19种不同的木糖还原酶(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+或NADP+之间的相互作用,并从中选择突变子K270R和K270N进行实验验证.突变基因及野生基因用异丙基-β-d-硫代半乳糖苷(IPTG)在大肠杆菌内进行诱导表达,经纯化后进行酶学性质研究.结果发现:K270R突变使得XR与NADP+的结合能力降低,米氏常数Km由0.025mmol/L升高到0.050mmol/L;K270N突变使得XR与NADP+不能结合.实验结果亦证实,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.

Lysine270 has been found to be a key amino acid that forms the binding pocket of PsXR （Piehia stipitis xylose reduetase） with nieotinamide adenine dinucleo,Lide phosphate （NADPH） or with nieotinamide adenine dinucleotide （NADH）. In order to investigate the effect of Lysine270 on the PsXR coenzyme specificity, 19 XR mu- tants were produced by substituting 19 amino acids tot Lysine270, and the interaction between XR mutants and NAD^＋ or NADP ~ was assessed by means of homology modeling and molecular docking. Then, K270R and K270N mutants were chosen to perform the bioinformatic analysis. After being induced by isopropyhhio-β-d-galactoside （IPTG） in Escherichia coli （E. coli）, the xylose reduetases of wild type and mutagenesis were purified and finally used to investigate the enzymatic properties. The results show that （ 1 ） K270R mutagenesis reduces the binding capability of XR with NADP^＋ and results in an increase of Michaelis constant from 0. 025 mmol/L to 0. 050 mmol/L; （2） K270N mutagenesis makes XR bind with NAD^＋ only; and （3） the coenzyme dependence of rationally-designed K270N completely reverses from NADPH to NADH.