Oxidoreductases are key enzymes in biocatalysis, but their dependence on the cofactor nicotinamide adenine dinucleotide (phosphate) (NAD(P)) presents challenges due to its high consumption and associated costs. Improving the cofactor utilization efficiency of these enzymes in biocatalysis is of great significance.

Enzyme fusion is a commonly used cofactor regeneration strategy, but the high usage and limited cofactor recovery restrict the sustainability of the catalytic process. Thus, there is an urgent need to develop methods to reduce the usage of NAD(P) to promote the efficiency of oxidoreductase-catalyzed processes.

A research team led by Prof. Yan Sun (Tianjin University) designed a peptide-bridged fusion oxidoreductase with electrostatic cofactor channeling, reducing NADPH input by two orders of magnitude or decreasing reaction time three-fold with the same cofactor input.

The results were published in the Chinese Journal of Catalysis.

A peptide bridging strategy was developed for linking phenylacetone monooxygenase and phosphite dehydrogenase to create nicotinamide adenine dinucleotide phosphate (NADP) channeling across the bridge by molecular dynamics (MD) simulations and experimental validations.

A decapeptide linker, R10 (RRRQRRRARR), has been identified as the most effective one. R10 linking fusion enzyme (FuE-R10) exhibits higher conversions than the mixed free enzyme system (MFEc) and the flexible peptide linked fusion enzyme, FuE-GS10, at a low NADPH/enzyme ratio (0.1).

Furthermore, FuE-R10 demonstrated significantly increased transportation effectiveness factors compared to other FuEs, indicating restricted NADP diffusion and efficient transportation between the enzymes’ NADP–binding pockets.

MD simulations revealed the positive value of the dissociation energy barrier for NADP in FuE-R10, proving the establishment of a cofactor channeling across the peptide.

Competitive side reaction experiment presented the effectiveness of FuE-R10 in suppressing side oxidizing reaction on NADPH, further affirming the presence of cofactor channeling in FuE-R10.

The electrostatic cofactor channeling was further verified by investigating the effect of ionic strength on the cascade reactions. Moreover, shortening the peptide bridge to five arginine residues (FuE-R5) further enhanced the channeling effect, as demonstrated by increased suppression of side oxidizing reaction, and increased cascade conversion compared to FuE-R10.

Remarkably, at 1 μmol L^–1 NADPH, 5 μmol L^–1 FuE-R5 functioned as 5 μmol L^–1 MFEc at 150 μmol L^–1 NADPH in ester synthesis. It implies that the cofactor input can be decreased by two orders of magnitude (to 1/150) by using FuE-R5 instead of MFEc, and the fusion enzyme can efficiently work at a sub-stoichiometric NADP concentration relative to the fusion enzyme.

This study has thus opened a new avenue to the development of cofactor channeling cascade biocatalysis for efficient and sustainable cofactor utilization.