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

• Published in: Enzyme and microbial technology 2020-03, Vol.134, p.109483, Article 109483
• Main Authors: Li, Yuanyuan, Pan, Hongyan, Chang, Yaowen, Dong, Na, Zou, Lei, Liang, Ping, Tian, Wei, Chang, Zunxue
• Format: Article
• Language: English
• Subjects: Capsella rubella steroid 5β-reductase; Cofactor specificity; Rational design; Site-directed mutagenesis
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2019.109483

•A steroid 5β-reductase in Capsella rubella and the key amino acid residues determing the cofactor specificity were identified and characterized.•CrSt5βR1 is a NADPH-dependent reductase and is capable of enantioselectively reducing the CC bond of various steroids and enones.•The R63K mutation enables the enzyme to use NADH as cofactor and the activity was further improved by the R64H mutation.•In combination with NADH regeneration systems CrSt5βR1 mutants are expected to have potential applications in pharmaceutical industry. Progesterone 5β-reductases (P5βRs) are involved in 5β-cardenolide formation by stereo-specific reduction of the △4,5 double bond of steroid precursors. In this study a steroid 5β-reductase was identified in Capsella rubella (CrSt5βR1) and its function in steroid 5β-reduction was validated experimentally. CrSt5βR1 is capable of enantioselectively reducing the activated CC 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 5β-pregnane-3,20-dione with NADH as cofactor, whereas the wild-type CrSt5βR1 is strictly NADPH-dependent. By further introducing the R64H mutation, the double mutant R63K_R64H of CrSt5βR1 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, AtP5βR and DlP5βR. CrSt5βR1 mutants are expected to have the potential for biotechnological applications in combination with the well-established NADH regeneration systems.