Switching the substrate specificity from NADH to NADPH by a single mutation of NADH oxidase from Lactobacillus rhamnosus.

Enzymatic NADP+ regeneration is a promising approach to produce valuable chemicals under economic conditions. Among all the enzymatic routes, using water-forming NADH oxidase is an ideal one because there is no by-product. However, most NADH oxidases have a low specific activity to NADPH. In this work, a thermostable NADH oxidase from Lactobacillus rhamnosus (LrNox) was rationally engineered to switch its specificity from NADH to NADPH. The results show that mutants D177A, G178R, D177A/G178R, D177A/G178R/L179S improved the NADPH activity by a factor of 4-6. The highest NADPH catalytic efficiency (Kcat/Km 223.71 S-1 μm-1, 47.6-fold higher than wild-type LrNox) and 51% of NADH activity retention were achieved by replacing the single amino acid Leu179 for serine (L179S) in LrNox. Modeling of L179S-NADPH complex reveals that the phosphate group of NADPH interacts with the hydroxyl of Ser179 with a strong hydrogen bond and several shorter hydrogen bonds with the amino group of Lys185 could stabilize the binding of NADPH in the L179S mutant. This work provides an efficient method for converting NAD(P)H specificity and shows that L179S mutant is a potential and efficient auxiliary enzyme for NADP+ regeneration.