Structural and mechanistic basis for the heterotetrameric benzaldehyde synthase from petunia.

Abstract

Benzaldehyde is a widespread volatile compound produced by plants. Its final biosynthetic step is catalyzed by benzaldehyde synthase (BS), a peroxisomal enzyme composed of α and β subunits, both belonging to the short-chain dehydrogenase/reductase (SDR) family. Here, we report the crystal structure of <i>Petunia hybrida</i> BS, which reveals an α₂β₂ heterotetrameric arrangement. Structural and biochemical analyses show that the α subunits contain the canonical catalytic site, whereas the β subunits have lost catalytic activity but are essential for heterotetramer assembly. Notably, the C terminus of the β subunit extends into the diagonally positioned α 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.