电子传递链的适配提高孕酮17α羟基化生物催化效率

Adaptation of the electron transport chain improves the biocatalytic efficiency of progesterone 17α hydroxylation

摘要:

17α羟化酶是转化孕酮制备各种孕激素药物中间体的关键酶。为提高该酶在生物催化中的特异性羟基化能力，本研究将来源于纤维素黏性细菌(Sorangium cellulosum) Soce56的羟化酶CYP260A1与大肠杆菌(Escherichia coli) K-12来源的Fpr和牛肾上腺来源的Adx_4-108组建成新的电子传递系统，用于孕酮的生物转化。通过对CYP260A1进行选择性突变，获得17α羟化酶活性显著提高的突变体S276I，经体外催化体系的优化设计，使17α-OH孕酮的产率达到58%。此外，利用定点突变技术探究铁氧还蛋白Adx_4-108的模拟磷酸化对17α羟化酶活性的影响，结果显示，突变体Adx_4-108T69E向S276I传递电子，进一步增强了对孕酮C17位的特异性，17α-OH孕酮的产率最终提高到74%。本研究为细菌来源的17α羟化酶特异性转化生产17α-OH孕酮提供了新的方案，为孕激素类药物在工业上利用生物转化法生产奠定了理论基础。

关键词:17α羟化酶;孕酮;电子传递链;蛋白突变

Abstract:

17α hydroxylase is a key enzyme for the conversion of progesterone to prepare various progestational drug intermediates. To improve the specific hydroxylation capability of this enzyme in steroid biocatalysis, the CYP260A1 derived from cellulose-mucilaginous bacteria Sorangium cellulosum Soce56 and the Fpr and bovine adrenal-derived Adx_4-108 derived from Escherichia coli str. K-12 were used to construct a new electron transfer system for the conversion of progesterone. Selective mutation of CYP260A1 resulted in a mutant S276I with significantly enhanced 17α hydroxylase activity, and the yield of 17α-OH progesterone reached 58% after optimization of the catalytic system in vitro. In addition, the effect of phosphorylation of the ferredoxin Adx_4-108 on 17α hydroxyl activity was evaluated using a targeted mutation technique, and the results showed that the mutation Adx_4-108T69E transferred electrons to S276I more efficiently, which further enhanced the catalytic specificity in the C17 position of progesterone, and the yield of 17α-OH progesterone was eventually increased to 74%. This study provides a new option for the production of 17α-OH progesterone by specific transformation of bacterial-derived 17α hydroxylase, and lays a theoretical foundation for the industrial production of progesterone analogs using biotransformation method.

Key words:17α hydroxylase;progesterone;electronic transmission chain;protein mutation