T4T7 Decoding Synthetic Biology 2023-07-18 18:45 Published in Shanghai

In the current pharmaceutical industry, enzyme catalyzed redox reactions play a crucial role. As the most commonly used hydrogen donors, nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) account for almost 90% of the biocatalytic redox process. However, the majority of wild nicotinamide adenine dinucleotide dehydrogenase (FDH) relies on NAD, and its catalytic activity is poor compared to other enzyme tools such as glucose dehydrogenase. Therefore, in order to achieve a reliable and robust NADPH regeneration system, it is necessary to develop FDH that is efficient and capable of utilizing NADP.

Recently, Xu Jian and his team from East China University of Science and Technology published a research paper titled "Engineering a Form Dehydrogenase for NADPH Regeneration" in ChemBioChem magazine, reporting a new form dehydrogenase (CdFDH) from Candida albicans. Through structurally guided rational design, CdFDH did not exhibit strict NAD preference and had good NADP utilization ability, Capable of supporting biocatalytic reactions with different NADPH dependencies.

Figure 1. Protein structure and mutation evolution path of CdFDH

In previous studies, the team successfully modified an NADP dependent mutant (BstFDH-G146M/A287G) from Burkholderia statis through rational design. However, further random mutations failed to obtain mutants with higher catalytic activity. Therefore, researchers turned to other FDHs to find better starting points for protein engineering. As a new member of NADP dependent FDH, CdFDH has become an ideal research object. Through structurally guided rational/semi rational design, CdFDH did not exhibit strict NAD dependence, and in the constructed combination mutant CdFDH-M4 (D197Q/Y198R/Q199N/A372S/K371T/∆ Q375/K167R/H16L/K159R), its catalytic efficiency (kcat/Km) for NADP was increased by 75 times. CdFDH-M4 exhibits excellent performance in various asymmetric redox processes, with its total coenzyme number of turns (TTN) ranging from 135 to 986, giving it the potential to be used in biocatalytic reactions that require NADPH.

Figure 2. Comparison of cofactor binding environments between wild-type and mutant M4

Afterwards, the team successfully obtained the optimal mutant CdFDH-M4 and NADP co crystallization (PDB: 8J3P) with a resolution of 2.3 Å. Research has found that compared to the wild-type, the binding environment of nicotinamide coenzyme has undergone significant changes, resulting in better NADP adaptability. Mutation sites D197Q, Y198R, and Q199N, CdFDH-M4 provide greater space near the hydroxyl group of NAD (H) ribose to accommodate phosphate groups. Mutation sites Gln197, Arg198, and Asn199 residues also form a hydrogen bond network with the phosphate group of NAD (P) H. In addition, the mutation site A372S introduces a novel hydrogen bonding network (Figure 2), and the phosphate group of Lys3 from another subunit supports additional polar contact with NAD (P) H, which was not observed in wild-type enzymes.

In summary, this study successfully modified an efficient NADP dependent FDH mutant (CdFDH-M4), providing strong support for the regeneration of nicotinamide coenzyme. CdFDH-M4 has good NADP utilization ability and can support different types of NADPH dependent biocatalytic reactions. Structural analysis and molecular dynamics studies have revealed the key role of mutation sites in promoting substrate/coenzyme binding and FDH catalyzed cyclic transformation. At the same time, the study also provides valuable examples for expanding the NADPH regeneration system and provides inspiration for improving the cofactor preference of the FDH superfamily.