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基于同源模型的比较和分析,发现羰基还原酶SCR1辅酶结合域P124和W125位点对辅酶NADPH的结合形成了一定的空间位阻效应.通过对该位点进行小侧基氨基酸的取代突变,该酶的底物专一性和立体选择性均发生了不同程度的改变,表明该位点是酶与辅酶有效结合的关键位点,而且它与辅酶结合的空间效应进一步影响了底物结合域活性中心对不同构型的底物及其对映体产物的亲和作用.在底物专一性方面,野生型酶对2-羟基苯乙酮和2-溴苯乙酮及其衍生物等底物表现出较高的催化活性,而突变株W125A,W125G,P124A/W125A和P124G/W125G对苯乙酮及其部分衍生物和2-辛酮等底物的催化活性均有所提高.对于酶的立体选择性,部分突变株发生了转化产物对映体构型反转的现象,突变株P124A/W125A和P124G/W125G催化还原2-羟基苯乙酮和4-氯乙酰乙酸乙酯均生成了(R)-型产物.
Based on the comparison and analysis of homology models, it was found that the binding sites of P124 and W125, a coenzyme binding domain of carbonyl reductase, formed a steric hindrance effect on the coenzyme NADPH binding.A small substitution of amino acid residues in this site, The substrate specificity and stereoselectivity of the enzyme all changed in varying degrees, indicating that the site is the key enzyme and coenzyme efficient binding sites, and its coenzyme with the spatial effect of further affecting the substrate binding domain Active centers on the different configurations of the substrate and its enantiomeric affinity of the product.In substrate specificity, the wild-type enzyme 2-hydroxyacetophenone and 2-bromoacetophenone and its derivatives, etc. The substrates showed higher catalytic activity, and the catalytic activity of mutants W125A, W125G, P124A / W125A and P124G / W125G on the substrates such as acetophenone and its derivatives and 2-octanone increased. Stereoselectivity of the enzyme, some of the mutant strains occurred inversion of the enantiomeric configuration of the conversion products, mutant strains P124A / W125A and P124G / W125G catalytic reduction 2-hydroxyacetophenone and ethyl 4-chloroacetoacetate are generated (R) -type product.