Rational Engineering of 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase for a Biomimetic Nicotinamide Mononucleotide Cofactor

Abstract

Enzymes are powerful biological catalysts for natural substrates but they have low catalytic efficiency for non-natural substrates. Protein engineering can be used to optimize enzymes for catalysis and stability. 3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidoreduction reaction of NAD+ with androsterone. Based on the structure and catalytic mechanism, we mutated the residues of T11, I13, D41, A70, and I112 and they interacted with different portions of NAD+ to switch cofactor specificity to biomimetic cofactor nicotinamide mononucleotide (NMN+). Compared to wild-type 3α-HSD/CR, the catalytic efficiency of these mutants for NAD+ decreased significantly except for the T11 mutants but changed slightly for NMN+ except for the A70K mutant. The A70K mutant increased the catalytic efficiency for NMN+ by 8.7-fold, concomitant with a significant decrease in NAD+ by 1.4 × 104-fold, resulting in 9.6 × 104-fold cofactor specificity switch toward NMN+ over NAD+. Meanwhile, the I112K variant increased the thermal stability and changed to a three-state transition from a two-state transition of thermal unfolding of wild-type 3α-HSD/CR by differential scanning fluorimetry. Molecular docking analysis indicated that mutations on these residues affect the position and conformation of the docked NAD+ and NMN+, thereby affecting their activity. A70K variant sterically blocks the binding with NAD+, restores the H-bonding interactions of catalytic residues of Y155 and K159 with NMN+, and enhances the catalytic efficiency for NMN+.