Poster: Secondary Metabolism
Abs #
288: Kinetic mechanism for successive hydroxylation by 5-epi-aristolochene-1,3-dihydroxylase
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Presenter: |
Shunji, Takahashi , shunjiwbb@yahoo.com |
Authors | Shunji, Takahashi (A) Bryan, Greenhagen T. (A) Yuxin, Zhao (B) Robert, Coates M. (B) Joe, Chappell (A) | | Affiliations: |
(A): University of Kentucky, Department of Agronomy
(B): University of Illinois, Department of Chemistry
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Capsidiol is a bicyclic, dihydroxylated sesquiterpene produced by many solanaceous plants in response to pathogen or elicitor-challenge and several other exogenous stimuli. Capsidiol prevents the germination and growth of several fungal species and is considered an important plant defense response. The biosynthesis of capsidiol is regulated by the expression of two key enzymes, a terpene synthase and a dihydroxylating P450 enzyme, 5-epi-aristolochene dihydroxylase (EAH). The latter enzyme is particularly intriguing because it catalyzes the stereo- and regio-specific addition of two hydroxyl functions to the sesquiterpene ring skeleton. To investigate the mechanistic features of EAH, detailed kinetic studies were used to determine the preferred order for hydroxylation, then 3-D homology modeling with the mammalian P450 2C5 structure was used to identify amino acid residues affecting the EAH hydroxylation activity. Detailed kinetic studies demonstrated that at high substrate (5-epi-aristolochene) concentrations, 3-deoxycapsidiol but not 1-deoxycapsidiol was produced. This was consistent with the catalytic efficiency (kcat/Km) of EAH for the 2 possible mono-hydroxylate intermediates, 1-deoxycapsidiol and 3-deoxycapsidiol, which were 0.031 and 0.34 mM-1 s-1. Additional confirmation that EAH catalyzes the conversion of 5-epi-aristolochene to 3-deoxycapsidiol, then to capsidiol was obtained by mutagenesis of several amino acid residues predicted to position 3-deoxycapsidiol in the active site for the second hydroxylation reaction. Mutation of serine 368 to cysteine, for example, resulted in a hydroxylase with near wildtype enzyme activity that only catalyzed the production of 3-deoxycapsidiol but no capsidiol. A structure-function model for the EAH enzyme will be presented.
