Dual factors required for cytochrome-P450-mediated hydrocarbon ring contraction in bacterial gibberellin phytohormone biosynthesis

Cytochromes P450 (CYPs) are heme-thiolate monooxygenases that prototypically cata-lyze the insertion of oxygen into unactivated C-H bonds but are capable of mediating more complex reactions. One of the most remarked -upon alternative reactions occurs during biosynthesis of the gibberellin A (GA) phytohormones, involving hydrocarbon ring contraction with coupled aldehyde extrusion of ent-kaurenoic acid to form the first gibberellin intermediate. While the unusual nature of this reaction has long been noted, its mechanistic basis has remained opaque. Building on identification of the relevant CYP114 from bacterial GA biosynthesis, detailed structure-function studies are reported here, including development of in vitro assays as well as crystallographic analyses both in the absence and presence of substrate. These structures provided insight into enzymatic catalysis of this unusual reaction, as exemplified by identification of a key role for the missing acid from an otherwise highly conserved acid-alcohol pair of residues. Notably, the results demonstrate that ring contraction requires dual factors, both the use of a dedicated ferredoxin and absence of the otherwise conserved acidic residue, with exclusion of either limiting turnover to just the initiating and more straightforward hydroxylation. The results provide detailed insight into the enzymatic structure-function relationships underlying this fascinating reaction and support the use of a semipinacol mechanism for the unusual ring contraction reaction.

Automated summary

Cytochromes P450 (CYPs) are enzymes that catalyze the insertion of oxygen into C-H bonds. One interesting reaction they can mediate is the biosynthesis of gibberellin A (GA) phytohormones, involving ring contraction and aldehyde extrusion. A recent study investigated the structure and function of the CYP114 enzyme involved in this reaction and found that it requires a dedicated ferredoxin and the absence of a conserved acidic residue. The results provide insight into the mechanism underlying this unusual reaction.