Cloning, heterologous expression, and functional characterization of 5-epi-Aristolochene-1, 3-Dihydroxylase from tobacco (Nicotiana tabacum)

Lyle Ralston, Soon Tae Kwon, Mark Schoenbeck, Jennifer Ralston, Joe Chappell, David J. Schenk, Robert M. Coates

Research output: Contribution to journalArticlepeer-review

112 Scopus citations


Capsidiol is a bicyclic, dihydroxylated sesquiterpene produced by several solanaceous species in response to a variety of environmental stimuli. It is the primary antimicrobial compound produced by Nicotiana tabacum in response to fungal elicitation, and it is formed via the isoprenoid pathway from 5-epi-aristolochene. Much of the biosynthetic pathway for the formation of this compound has been elucidated, except for the enzyme(s) responsible for the conversion of 5-epi-aristolochene to its dihydroxylated form, capsidiol. Biochemical evidence from previous studies with N. tabacum (Whitehead, I. M., Threlfall, D. R., and Ewing, D. F., 1989, Phytochemistry 28, 775-779) and Capsicum annuum Hoshino, T., Yamaura, T., Imaishi, H., Chida, M., Yoshizawa, Y., Higashi, K., Ohkawa, H., Mizutani, J., 1995, Phytochemistry 38, 609-613. suggested that the oxidation of 5-epi-aristolochene to capsidiol was mediated by at least one elicitor-inducible cytochrome P450 hydroxylase. In extending these observations, we developed an in vivo assay for 5-epi-aristolochene hydroxylase activity and used it to demonstrate a dose-dependent inhibition of activity by ancymidol and ketoconazole, two well characterized inhibitors of cytochrome P450 enzymes. Using degenerate oligonucleotide primers designed to the well conserved domains found within most P450 enzymes, including the heine binding domain, cDNA fragments representing four distinct P450 families (CYP71, CYP73, CYP82, and CYP92) were amplified from a cDNA library prepared against mRNA from elicitor-treated cells using PCR. The PCR fragments were subsequently used to isolate full-length cDNAs (CYP71D20 and D21, CYP73A27 and A28, CYP82E1 and CYP92A5), and these in turn were used to demonstrate that thee corresponding mRNAs were all induced in elicitor-treated cells, albeit with different induction patterns. Representative, full-length cDNAs for each of the P450s were engineered into a yeast expression system, and the recombinant yeast assessed for functional expression of P450 protein by measuring the CO difference spectra of the yeast microsomes. Only microsomal preparations from yeast expressing the CYP71D20 and CYP92A5 cDNAs exhibited significant CO difference absorbance spectra at 450 nm and were thus tested for their ability to hydroxylate 5-epi-aristolochene and 1-deoxycapsidiol, a putative mono-hydroxylated intermediate in capsidiol biosynthesis. Interestingly, the CYP71D20-encoded enzyme activity was capable of converting both 5-epi-aristolochene and 1-deoxycapsidiol to capsidiol in vitro, consistent with the notion that this P450 enzyme catalyzes both hydroxylations of its hydrocarbon substrate.

Original languageEnglish
Pages (from-to)222-235
Number of pages14
JournalArchives of Biochemistry and Biophysics
Issue number2
StatePublished - Sep 15 2001

Bibliographical note

Funding Information:
This work was supported by the Kentucky Agricultural Experiment Station and grants from NSF (J.C.) and NIH (R.M.C.). We thank Drs. Michael Barrett and Daniel Werck-Reichhart for helpful suggestions during the course of this work, Dr. Jack Goodman for his valuable assistance with the GC–MS analysis, and Dr. David Nelson for assigning the cytochrome nomenclature for the enzymes described here.


  • 5-epi-aristolochene hydroxylase
  • Cytochrome P450
  • Phytoalexin
  • Sesquiterpene cyclase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology


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