3.2 Constructing the NADPH cycle with the modification ofPdPDH
There is no NADP+-dependent galactitol dehydrogenase in the ENZYME database, and it is difficult to mine the NADP+-dependent galactitol dehydrogenase in nature. Therefore, we must develop a rational design for NADP+-dependent galactitol dehydrogenase. We minedPd PDH and Rl GDH from the enzyme bank, and their dehydrogenase activities were compared (Figure S2A). The activity ofPd PDH with respect to galactitol dehydrogenation was 17.29 times greater than that of Rl GDH (25.42 and 1.47 U mg−1, respectively) (Figure S2B). Therefore,Pd PDH was selected as the template for the rational design.
We docked Pd PDH (PDBID: 7E6O) with the cofactor NAD+ (PubChem CID: 5892) using the Schrodinger software, and the docking results are shown using LigPlot software (Figure S3A). We selected 22 amino acids within 5 Å of NAD+ that interact with the cofactor based on hydrogen bond or hydrophobic interaction. The Pd PDH-NAD+combination model was predicted using the CSR-SALAD website (Table S3 and Table S4). According to the predicted results, we designed 100 mutants of Pd PDH (Table S5) based on 13 amino acid residues (Figure S3B). Subsequently, a point mutation of Pd PDH was simulated using Schrodinger software, and the mutants were docked with the cofactor NADP+. Then, the docking binding energies of the mutants were counted (Table S5). A total of 14 mutants were selected according to their lower binding energies, indicating that they had a higher affinity for NADP+ than the other mutants. Furthermore, their specific activities for NAD+ and NADP+ were measured. As shown in Table 1, nine mutants completely reversed the cofactor preference and exhibited improved specific activity toward NADP+. Six mutants had NADP+ activities of more than 15 U mg−1; particularly, the NADP+activity of the D36A/I37R mutant reached 19.4 U mg−1. However, the triple mutants (A14T/D36A/I37R and A14S/D36A/I37R) did not improve their activities further than those of the double mutants. The single mutants (D36A, D36G, and I37R) exhibited no activity against NADP+. The screening accuracy was increased sevenfold the computer-aided screening (9/14) compared with the original screening (9/100).
The kinetic parameters of Pd PDH mutants and the wild-type are shown in Table 2. TheKm value of NADP+ with the D36A/I37R mutant was 0.942 mM and was lower than that of the wild-type enzyme (infinite), proving that there was a substantial increase in the affinity of the D36A/I37R mutant for NADP+. Moreover, the specificity constant (kcat/Km ) of the D36A/I37R mutant toward NADP+ reached 0.322 mM−1s−1, whereas that of the wild-type was 0 mM−1s−1. Conversely, the specificity constant (kcat/Km ) of the wild-type toward NAD+ was 0.4872 mM−1s−1, whereas that of the D36A/I37R mutant was 0 mM−1s−1. Furthermore, we identified the binding free energy of each cofactor with the wild-type and its mutant D36A/I37R (Table S6). The binding free energy of the D36A/I37R mutant toward NADP+ decreased by 54% (from −8.011 to −12.335 kcal mol−1), whereas that toward NAD+ increased by 46% (from −13.614 to −7.325 kcal mol−1). These results suggested that the binding affinity of the D36A/I37R mutant toward NADP+ was higher than that toward NAD+. Therefore, we obtained an NADP+-dependentPd PDHD36A/I37R for performing the NADPH cycle during tagatose production.