KO (kaurene oxidase) is a multifunctional cytochrome P450 catalysing three sequential oxidations in gibberellin phytohormone biosynthesis. These serve to transform the C4α 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 18O2 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 C4α 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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Research Article|
October 11 2010
Characterization of the kaurene oxidase CYP701A3, a multifunctional cytochrome P450 from gibberellin biosynthesis Available to Purchase
Dana Morrone;
Dana Morrone
*Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, U.S.A.
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Xiaoming Chen;
Xiaoming Chen
1
*Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, U.S.A.
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Robert M. Coates;
Robert M. Coates
†Department of Chemistry, University of Illinois, Urbana, IL 61801, U.S.A.
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Reuben J. Peters
Reuben J. Peters
2
*Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, U.S.A.
2To whom correspondence should be addressed (email [email protected]).
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Publisher: Portland Press Ltd
Received:
April 19 2010
Revision Received:
August 06 2010
Accepted:
August 11 2010
Accepted Manuscript online:
August 11 2010
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2010 Biochemical Society
2010
Biochem J (2010) 431 (3): 337–347.
Article history
Received:
April 19 2010
Revision Received:
August 06 2010
Accepted:
August 11 2010
Accepted Manuscript online:
August 11 2010
Citation
Dana Morrone, Xiaoming Chen, Robert M. Coates, Reuben J. Peters; Characterization of the kaurene oxidase CYP701A3, a multifunctional cytochrome P450 from gibberellin biosynthesis. Biochem J 1 November 2010; 431 (3): 337–347. doi: https://doi.org/10.1042/BJ20100597
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