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.
