3-甾酮-9α-羟基化酶的C-1，2位脱氢和C-9位羟基化双重特性的研究

Dehydrogenation at C-1,2 and Hydroxylation at C-9 by 3-Ketosteroid-9α-hydroxylase

摘要:

目的 验证来自新金分枝杆菌（Mycobacterium neoaurum) NRRL B-3805的3-甾酮-9α-羟基化酶（KSH）是否具有C-9位羟基化和C-1，2位脱氢功能。方法 将ksh基因中的加氧酶组分kshA395和还原酶组分kshB122分别在大肠杆菌（Escherichia coli, E. coli) BL21（DE3）中进行异源表达，以4-雄烯-3，17-二酮（4-AD）为底物，分别用KshA395、KshB122以及两者等比例混合对其进行酶催化反应。通过对反应体系优化构建pET28a-kshB-kshA等融合质粒，对KSH的双组分酶进行共表达，并以4-AD为底物进行酶催化反应。结果 KshA395和KshB122均对4-AD表现出脱氢功能，产物为1，4-雄二烯-3，17-二酮（ADD），未出现羟基化作用；等比例混合的KSH双组分酶在催化4-AD过程中，前期表现出强烈的C-9位羟基化作用，后期羟基化功能关闭，转为C-1，2位脱氢功能。KSH的双组分融合蛋白，包括KshB122-MBP-KshA395、MBP-KshB122-MBP-KshA395、MBP-KshB122-KshA395，在催化4-AD前期表现出C-9位羟基化作用，后期转变为C-1，2位脱氢作用；而KshB122-KshA395仅表现出C-1，2位脱氢作用，未表现出羟基化作用。结论 在甾醇降解途径中，KSH可以同时承担羟基化及脱氢功能，且先在C-9位羟基化，随后在C-1，2位脱氢，导致甾醇母核B环打开。

Abstract:

Objective The aim of the study is to verify the roles of 3-ketosteroid-9α-hydroxylase (KSH) from Mycobacterium neoaurum NRRL B-3805 in hydroxylation at C-9 and dehydrogenation at C-1,2.Method The oxygenase component kshA395 and the reductase component kshB122 from ksh were heterologously expressed in Escherichia coli (E. coli) BL21(DE3) respectively. With 4-androstene-3,17-dione (4-AD) as substrate, the enzyme-catalyzed reactions were carried out with KshA395, KshB122, and mixture of KshA395 and KshB122 in a ratio of 1∶1. Furthermore, the reaction system was optimized, and fusion plasmids such as pET28a-kshB-kshA were constructed for co-expression of the two-component enzyme of KSH. In addition, the enzyme-catalyzed reaction was performed with 4-AD as the substrate.Result Both KshA395 and KshB122 showed dehydrogenation on 4-AD, and the product was 1,4-androstene-3,17-dione (ADD), with no hydroxylation. The two-component enzyme of KSH, which was mixed in a ratio of 1∶1, showed strong hydroxylation at C-9 in phase 1 of the reaction. In phase 2, hydroxylation was shut down, and dehydrogenation at C-1,2 of 4-AD was turned on. The fusion proteins of KSH, such as KshB122-MBP-KshA395, MBP-KshB122-MBP-KshA395, and MBP-KshB122-KshA395, showed hydroxylation at C-9 in phase 1 and dehydrogenation at C-1,2 in phase 2 of the reaction. Nevertheless, KshB122-KshA395 showed only dehydrogenation at C-1,2 and no hydroxylation.Conclusion In the sterol degradation pathway, KSH can play a role in both hydroxylation and dehydrogenation. It first plays a role in hydroxylation at C-9 and then dehydrogenation at C-1,2, which leads to the degradation of the ring-B in steroids.