Solavetivone, a potent antifungal phytoalexin, is derived from a vetispirane-type sesquiterpene, premnaspirodiene, by a putative regio- and stereo-specific hydroxylation, followed by a second oxidation to yield the α,β-unsaturated ketone. Mechanistically, these reactions could occur via a single, multifunctional cytochrome P450 or some combination of cytochrome P450s and a dehydrogenase. We report here the characterization of a single cytochrome P450 enzyme, Hyoscyamus muticus premnaspirodiene oxygenase (HPO), that catalyzes these successive reactions at carbon 2 (C-2) of the spirane substrate. HPO also catalyzes the equivalent regio-specific (C-2) hydroxylation of several eremophilane-type (decalin ring system) sesquiterpenes, such as with 5-epi-aristolochene. Moreover, HPO displays interesting comparisons to other sesquiterpene hydroxylases. 5-Epi-aristolochene di-hydroxylase (EAH) differs catalytically from HPO by introducing hydroxyl groups first at C-1, then C-3 of 5-epi-aristolochene. HPO and EAH also differ from one another by 91-amino acid differences, with four of these differences mapping to putative substrate recognition regions 5 and 6. These four positions were mutagenized alone and in various combinations in both HPO and EAH and the mutant enzymes were characterized for changes in substrate selectivity, reaction product specificity, and kinetic properties. These mutations did not alter the regio- or stereo-specificity of either HPO or EAH, but specific combinations of the mutations did improve the catalytic efficiencies 10-15-fold. Molecular models and comparisons between HPO and EAH provide insights into the catalytic properties of these enzymes of specialized metabolism in plants.
|Number of pages||11|
|Journal||Journal of Biological Chemistry|
|State||Published - Oct 26 2007|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology