弗氏链霉菌泰乐菌素还原酶原核表达及结构功能研究

Study on the prokaryotic expression, structure, and function of tylosin reductase from Streptomyces fradiae

目的探究弗氏链霉菌中的泰乐菌素还原酶的结构和功能。方法通过NCBI数据库比对查找得到泰乐菌素还原酶蛋白序列及其基因序列,建立该蛋白三维模型和系统发育树,对酶蛋白进行原核表达及纯化,并测定其活性。结果来源于弗氏链霉菌ATCC19609菌株(Streptomyces fradiae ATCC19609)的泰乐菌素还原酶基因由996bp碱基组成,编码331个氨基酸残基。该酶三级结构包括10个桶状α螺旋,8个β折叠,一个还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)结合位点。系统发育分析表明其属于醛酮还原酶(Aldo-keto reductase,AKR)超家族的AKR12家族。酶活力测定结果表明,泰乐菌素还原酶具有宽泛的底物谱,对泰乐菌素比活力为0.128U/mg。结论证实了弗氏链霉菌泰乐菌素还原酶可以催化还原泰乐菌素,为抑制泰乐菌素还原酶活性,提高泰乐菌素发酵产品的纯度,提供了有效的理论依据。

Objective In this study, the structure and function of tylosin reductase from Streptomyces fradiae was explored. Methods The protein sequence and gene sequence of tylosin reductase were obtained by searches and comparisons in NCBI database, and the three-dimensional model and phylogenetic tree of tylosin reductase were established. In addition, prokaryotic expression and purification of the enzyme was performed and the activity of enzyme was determined. Results The sequence length of tylosin reductase gene from Streptomyces fradiae ATCC 19609 was 996 bp, which encoded a polypeptide of 331 amino acids. The tertiary structure of the enzyme included ten barrel-shaped α helices, eight β folds and a NADPH binding site. Phylogenetic analysis showed that tylosin reductase belonged to the AKR12 family of aldo-keto reductase superfamily. The results of determination of enzyme activity demonstrated tylosin reductase had a wide range of substrates, and its specific activity to tylosin was 0.128 U/mg.Conclusion It is confirmed that tylosin reductase from Streptomyces fradiae could catalyze the reduction of tylosin.This experiment can provide basic theoretical support for inhibiting the activity of tylosin reductase and improving the purity of tylosin fermentation products.