Kaurene oxidase (KO) is a multifunctional cytochrome P450 catalyzing three sequential oxidations in gibberellin phytohormone biosynthesis. These serve to transform the C4a methyl of the ent-kaurene olefin intermediate into the carboxylic acid moiety of ent-kauren-19-oic acid. To investigate the unknown catalytic mechanism and properties of KO, we have engineered the corresponding CYP701A3 from Arabidopsis thaliana (AtKO) for functional recombinant expression in E. coli, involving use of a fully codon-optimized construct, along with additional N-terminal deletion and modification. This recombinant AtKO (rAtKO) was used to carry out 18O2 labeling studies with ent-kaurene, and the intermediates ent-kaurenol and ent-kaurenal, to investigate the multifunctional reaction sequence; revealing catalysis of three hydroxylation reactions, which further requires dehydration at some stage. Accordingly, following initial hydroxylation, ent-kaurenol must then be further hydroxylated to a gem-diol intermediate, and our data indicates that the subsequent reactions proceed via dehydration of the gem-diol to ent-kaurenal, followed by an additional hydroxylation to directly form ent-kaurenoic acid. Kinetic analysis indicates that these intermediates are all retained in the active site during the course of the reaction series, with the first hydroxylation being rate limiting. In addition, investigation of alternative substrates demonstrated that ent-beyerene, which differs in ring structure distal to the C4a methyl, is only hydroxylated by rAtKO, indicating the importance of the exact tetracyclic ring structure of kaurane for multifunctional KO activity. Thus, the results presented here clarify the reaction sequence and enzymatic mechanism of KO, as well as substrate features critical for the catalyzed multiple reaction sequence.

Read the original article at BJ Plant.