A comparative study of two aldehyde dehydrogenases from Sphingobium sp.: the substrate spectrum and catalytic mechanism.

Biocatalytic oxidation is one of the most important and indispensable organic reactions for the development of green and sustainable biomanufacturing processes. NAD(P) ⁺ -dependent aldehyde dehydrogenase (ALDH) catalyzes the oxidation of aldehydes to carboxylic acids. Here, two ALDHs, Sp ALDH1 and Sp ALDH2, were identified from Sphingobium sp. SYK-6. They belong to different ALDH families and share only 32.30% amino acid identity. Interestingly, Sp ALDH1 and Sp ALDH2 exhibit significantly different enzymatic properties and substrate profiles. Sp ALDH2 has better thermostability than Sp ALDH1. Sp ALDH1 is a metalloenzyme and is activated by potassium ions, while Sp ALDH2 is not metallic-dependent. Compared with Sp ALDH1, Sp ALDH2 has a relatively broad substrate spectrum toward aromatic aldehydes. Based on homology modeling and molecular docking analysis, mechanisms underlying the substrate specificity of ALDHs were elucidated. For both ALDHs, hydrophobicity of substrate binding pockets is important for the catalytic properties, especially substrate specificity. Notably, optimization of the flexible loop 444-457 reforms a hydrogen bond between pyridine substrates and Sp ALDH1, contributing to the high catalytic activity. Finally, a coupling reaction catalyzed by ALDHs and NOX was constructed for efficient production of aromatic carboxylic acids.