Received August 2, 2001
Accepted November 1, 2001
Characterization of Phenylpropene O-Methyltransferases from Sweet Basil:
Facile Change of Substrate Specificity and Convergent Evolution within a Plant
O-Methyltransferase Family
David R. Gang 1*, Noa Lavid 2, Chloe Zubieta 3, Feng Chen 1, Till Beuerle 1, Efraim Lewinsohn 2, Joseph P. Noel 3, and Eran Pichersky 1
1
Department of Molecular, Cellular and Developmental Biology, University of Michigan,
Ann Arbor, Michigan 48109-1048
2
Aromatic, Medicinal and Spice Crops Unit, Newe Ya'ar Research Center, Agricultural
Research Organization, P.O. Box 1021, Ramat Yishay, 30095, Israel
3
Structural Biology Laboratory, The Salk Institute for Biological Studies, 10010 North
Torrey Pines Road, La Jolla, California 92037
* To whom correspondence should be addressed.
Some basil varieties are able to convert the phenylpropenes chavicol and eugenol
to methylchavicol and methyleugenol, respectively. Chavicol O-methyltransferase
(CVOMT) and eugenol O-methyltransferase (EOMT) cDNAs were isolated from
the sweet basil variety EMX-1 using a biochemical genomics approach. These cDNAs
encode proteins that are 90% identical to each other and very similar to several
isoflavone O-methyltransferases such as IOMT, which catalyzes the 4'-
O-methylation of 2,7,4'-trihydroxyisoflavanone. On the other hand,
CVOMT1 and EOMT1 are related only distantly to (iso)eugenol OMT from Clarkia
breweri, indicating that the eugenol O-methylating enzymes in basil
and C. breweri evolved independently. Transcripts for CVOMT1 and EOMT1 were
highly expressed in the peltate glandular trichomes on the surface of the young basil
leaves. The CVOMT1 and EOMT1 cDNAs were expressed in Escherichia
coli, and active proteins were produced. CVOMT1 catalyzed the O-methylation
of chavicol, and EOMT1 also catalyzed the O-methylation of chavicol with
equal efficiency to that of CVOMT1, but it was much more efficient in O-methylating
eugenol. Molecular modeling, based on the crystal structure of IOMT, suggested that
a single amino acid difference was responsible for the difference in substrate discrimination
between CVOMT1 and EOMT1. This prediction was confirmed by site-directed mutagenesis,
in which the appropriate mutants of CVOMT1 (F260S) and EOMT1 (S261F) were produced
that exhibited the opposite substrate preference relative to the respective native
enzyme.
