Bacillaene酮还原酶结构域的异源表达及底物特异性分析

Heterologous expression and substrate specificity of ketoreductase domain in bacillaene polyketide synthase

摘要:

Bacillaene生物合成过程中，聚酮合酶第一个延伸模块的酮还原酶结构域 (BacKR1) 既催化α酮基的还原，也催化β酮基的还原，具有天然的底物宽泛性。为进一步研究该结构域的底物特异性，在大肠杆菌中对其进行了异源表达。体外酶学分析表明BacKR1可以催化聚酮类底物 (±)-2-甲基-3-氧代戊酸-乙酰半胱胺硫酯外消旋体的立体选择性还原，仅生成4种非对映异构体中的一种，此外BacKR1还可以催化环己酮和对氯苯乙酮等非聚酮类底物的还原，暗示了聚酮合酶中酮还原酶结构域作为生物催化剂的潜力。

Abstract:

The ketoreductase (KR) domain in the first extending module of the polyketide synthase (PKS) catalyzes the reductions of both an a-keto group and a b-keto group in the biosynthesis of bacillaene, suggesting the intrinsic substrate promiscuity. In order to further investigate the substrate specificity, the KR domain (BacKR1) was heterologously overexpressed in Escherichia coli. In vitro enzymatic analysis showed that only one of the four diastereomers was formed in the reduction of the racemic (±)-2-methyl-3-oxopentanoyl-N-acetylcysteamine thioester catalyzed by BacKR1. In addition, BacKR1 was revealed to catalyze the reductions of cyclohexanone and p-chloroacetophenone, indicating the potential of KR domians of PKSs as biocatalysts.