Structural Superimposition

Abstact

Benzaldehyde is a widespread volatile compound produced by plants. Its final biosynthetic step is catalyzed by benzaldehyde synthase (BS), a peroxisomal enzyme composed of alpha and beta subunits, both belonging to the short-chain dehydrogenase/reductase (SDR) family. Here, we report the crystal structure of Petunia hybrida BS, which reveals an alpha(2)beta(2) heterotetrameric arrangement. Structural and biochemical analyses show that the alpha subunits contain the canonical catalytic site, whereas the beta subunits have lost catalytic activity but are essential for heterotetramer assembly. Notably, the C terminus of the beta subunit extends into the diagonally positioned alpha subunit, contributing to the formation of the composite benzoyl-CoA substrate-binding pocket. Site-directed mutagenesis and subunit-mixing experiments support noncooperative, additive contributions of protomers within the heterotetramer. This work establishes BS as a rare heterotetrameric plant SDR and demonstrates how subunit specialization and intersubunit arrangement enable function, providing principles for understanding and engineering multimeric enzyme complexes.