Kinetic and molecular analysis of 5-epiaristolochene 1,3-dihydroxylase, a cytochrome P450 enzyme catalyzing successive hydroxylations of sesquiterpenes

Shunji Takahashi, Yuxin Zhao, Paul E. O'Maille, Bryan T. Greenhagen, Joseph P. Noel, Robert M. Coates, Joe Chappell

Research output: Contribution to journalArticlepeer-review

37 Scopus citations

Abstract

The final step of capsidiol biosynthesis is catalyzed by 5-epiaristolochene dihydroxylase (EAH), a cytochrome P450 enzyme that catalyzes the regio- and stereospecific insertion of two hydrosyl moieties into the bicyclic sesquiterpene 5-epiaristolochene (EA). Detailed kinetic studies using EA and the two possible monohydroxylated intermediates demonstrated the release of 1β-hydroxy-EA ((OH)EA) at high EA concentrations and a 10-fold catalytic preference for 1β(OH)EA versus 3α(OH)EA, indicative of a preferred reaction order of hydrosylation at C-1, followed by that at C-3. Sequence alignments and homology modeling identified active-site residues tested for their contribution to substrate specificity and overall enzymatic activity. Mutants EAH-S368C and EAH-S368V exhibited wild-type catalytic efficiencies for 1β(OH)EA biosynthesis, but were devoid of the successive hydroxylation activity for capsidiol biosynthesis. In contrast to EAH-S368C, EAH-S368V catalyzed the relative equal biosynthesis of 1β(OH)EA, 2β(OH)EA, and 3β(OH)EA from EA with wild-type efficiency. Moreover, EAH-S368V converted ∼1.5% of these monohydroxylated products to their respective ketone forms. Alanine and threonine mutations at position 368 were significantly compromised in their conversion rates of EA to capsidiol and correlated with 3.6- and 5.7-fold increases in their Km values for the 1β(OH)EA intermediate, respectively. A role for Ile486 in the successive hydroxylations of EA was also suggested by the EAH-I468A mutant, which produced significant amounts 1β(OH)EA, but negligible amounts of capsidiol from EA. The altered product profile of the EAH-I486A mutant correlated with a 3.6-fold higher Km for EA and a 4.4-fold slower turnover rate (k cat) for 1β(OH)EA. These kinetic and mutational studies were correlated with substrate docking predictions to suggest how Ser368 and Ile486 might contribute to active-site topology, substrate binding, and substrate presentation to the oxo-Fe-heme reaction center.

Original languageEnglish
Pages (from-to)3686-3696
Number of pages11
JournalJournal of Biological Chemistry
Volume280
Issue number5
DOIs
StatePublished - Feb 4 2005

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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