Engineering and characterization of hybrid carboxylic acid reductases

Carboxylic acid reductases (CARs) are valuable biocatalysts due to their ability to reduce a broad range of carboxylate substrates into the corresponding aldehyde products. CARs are multi-domain enzymes with separate catalytic domains for the adenylation and the subsequent reduction of substrates. Inter-domain dynamics are crucial for the catalytic activities of CARs. In this work, hybrid enzymes that contain domains from four bacterial CARs and one fungal CAR were constructed based on domain boundaries that were defined using a combination of bioinformatics and structural analysis. Hybrid CARs were characterized in both steady-state and transient kinetics studies using aromatic and straight-chain (C₃-C₅) carboxylate substrates. Kinetic data support that the inter-domain interactions play an important role in the function of both wild-type and hybrid CARs and further lead to the hypothesis that reduction is the rate-determining step in CAR catalysis. Our results provide both fundamental insights into CAR catalysis and rationale for hybrid CAR engineering.