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[摘要]：KO (kaurene oxidase) is a multifunctional cytochrome P450 catalysing three sequential oxidations in gibberellin phytohormone biosynthesis These serve to transform the C4 alpha 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 Escherichia 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 O-18(2) labelling 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 indicate 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 of the present study clarify the reaction sequence and enzymatic mechanism of KO, as well as substrate features critical for the catalysed multiple reaction sequence |
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