Identification of key sites determining the cofactor specificity and improvement of catalytic activity of a steroid 5 beta-reductase from Capsella rubella

摘要

Progesterone 5 beta-reductases (P5 beta Rs) are involved in 5 beta-cardenolide formation by stereo-specific reduction of the Delta(4,5) double bond of steroid precursors. In this study a steroid 5 beta-reductase was identified in Capsella rubella (CrSt5 beta R1) and its function in steroid 5 beta-reduction was validated experimentally. CrSt5 beta R1 is capable of enantioselectively reducing the activated C=C bond of broad substrates such as steroids and enones by using NADPH as a cofactor and therefore has the potential as a biocatalyst in organic synthesis. However, for industrial purposes the cheaper NADH is the preferred cofactor. By applying rational design based on literature and complementary mutagenesis strategies, we successfully identified two key amino acid residues determining the cofactor specificity of the enzyme. The R63 K mutation enables the enzyme to convert progesterone to 5 beta-pregnane-3,20-dione with NADH as cofactor, whereas the wild-type CrSt5 beta R1 is strictly NADPH-dependent. By further introducing the R64H mutation, the double mutant R63K_R64H of CrSt5 beta 111 was shown to increase enzymatic activity by13.8-fold with NADH as a cofactor and to increase the NADH/NADPH conversion ratio by 10.9-fold over the R63 K single mutant. This finding was successfully applied to change the cofactor specificity and to improve activity of other members of the same enzyme family, AtP5 beta R and D/PSAR. CrSt5 beta R1 mutants are expected to have the potential for biotechnological applications in combination with the well-established NADH regeneration systems.