# High coenzyme affinity chimeric amine dehydrogenase based on domain engineering

## TL;DR

Scientists improved an enzyme's efficiency and stability by combining parts from different enzymes, making it better for industrial use.

## Contribution

A chimeric amine dehydrogenase with enhanced coenzyme affinity and catalytic efficiency was created through domain engineering.

## Key findings

- cFLF-AmDH showed a twofold improvement in NADH affinity and a 4.4-fold increase in catalytic efficiency compared to F-AmDH.
- The chimeric enzyme had a 60% longer half-life at 55°C and a broader substrate spectrum than F-AmDH.
- The reaction rate increased by 150% in reductive amination when using cFLF-AmDH at 0.05 mM NAD+.

## Abstract

NADH-dependent phenylalanine amine dehydrogenase (F-AmDH) engineered from phenylalanine dehydrogenase (PheDH) catalyzes the synthesis of aromatic chiral amines from prochiral ketone substrates. However, its low coenzyme affinity and catalytic efficiency limit its industrial application. Here, we developed a chimeric amine dehydrogenase, cFLF-AmDH, based on the relative independence of the structure at the domain level, combined with a substrate-binding domain from F-AmDH and a high-affinity cofactor-binding domain from leucine amine dehydrogenase (L-AmDH). The kinetic parameters indicated that cFLF-AmDH showed a twofold improvement in affinity for NADH and a 4.4-fold increase in catalytic efficiency (kcat/Km) compared with the parent F-AmDH. Meanwhile, cFLF-AmDH also showed higher thermal stability, with the half-life increased by 60% at 55 °C and a broader substrate spectrum, than the parent F-AmDH. Molecular dynamics simulations suggested that the constructed cFLF-AmDH had a more stable structure than the parent F-AmDH, thereby improving the affinity of the coenzyme. The reaction rate increased by 150% in the reductive amination reaction catalyzed by cFLF-AmDH. When the NAD+ concentration was 0.05 mM, the conversion rate was increased by 150%. These results suggest that the chimeric protein by domain shuffling from different domain donors not only increased the cofactor affinity and catalytic efficiency, but also changed the specificity and thermal stability. Our study highlights that domain engineering is another effective method for creating biodiversity with different catalytic properties.

The online version contains supplementary material available at 10.1186/s40643-022-00528-0.