Engineering glucose dehydrogenase from Sulfolobus sulfataricus toward utilizing diverse nicotinamide cofactor biomimetics

Oxidoreductases are crucial for the production of widely utilized chiral building blocks, giving rise to a demand for costly NAD(P)/H. As potential substitutes for natural cofactors, nicotinamide cofactor biomimetics (NCBs) have been extensively explored due to their cost-efficiency and easy synthesis. Ss GDH is the first enzyme identified to utilize oxidized NCBs, enabling NCBs-based in situ cofactor regeneration. However, Ss GDH remains limited in cofactor scope, and the catalytic efficiency is inadequate. Here, a series of synthetic NCBs were designed and synthesized. By modification of interface residues, Ss GDH was engineered for improved catalytic efficiency and adaptation toward diverse NCBs. V296I/H297G M2 showed a 47.2-fold increase in catalytic efficiency compared with WT when utilizing newly synthesized p -BNNA + (3-carbamoyl-1-(4-nitrobenzyl) pyridin-1-ium), demonstrating the importance of interface sites V296 and H297. Beneficial variants were coupled with XenA-catalyzed reaction to demonstrate their universality as NCBs regenerative enzymes. Based on all-atom molecular dynamic simulation, F279, a residue anchored by H297 in WT, was found to stabilize NCBs with more flexible conformation in V296I/H297G M2 . Furthermore, the widened cofactor entry tunnel is conducive to the entry and binding of NCBs. These results provide valuable insights into engineering and de novo design of enzymes for utilizing NCBs. Our study explored nicotinamide cofactor biomimetics (NCBs) as cost-effective alternatives for natural cofactors. Ss GDH was engineered toward utilizing newly designed NCBs. The catalytic mechanism was elucidated by all-atom MD simulations, contributing to the development of biocatalytic processes in a more sustainable manner.