Characterization of phenylpropene O-methyltransferases from sweet basil: facile change of substrate specificity and convergent evolution within a plant O-methyltransferase family

Abstract

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.

Figure 1.

Role of Selected SMOMTs in Plant Specialized Metabolism. (A) Simplified biochemical pathway leading to methylchavicol and methyleugenol in sweet basil and to the methoxylated isoflavonoids 2,7-dihydroxy-4′-methoxyisoflavanone and pisatin in legumes. Transformations catalyzed by CVOMT, EOMT, IOMT, and HMOMT are indicated by single arrows. Double arrows indicate multiple conversions. (B) Selected conversions catalyzed by COMT.

Figure 2.

Members of the COMT Superfamily of Plant SMOMTs Fit into Two Classes Based on Sequence Homology. Class I contains COMTs and enzymes such as C. breweri IEMT, which evolved recently from this class. Class II contains OMTs that use a variety of substrate structures. Arabidopsis thaliana OMT does not fit into either class. The sequences of the genes with names in boldface are compared in Figure 3.

Figure 3.

Amino Acid Sequence Alignment Comparing Selected Members of the COMT Superfamily of Plant OMTs.

Figure 4.

Tissue-Specific Expression of Basil Phenylpropene OMTs. (A) RNA gel blot analysis of gene expression. Lanes 1 to 4 (3 μg of total RNA per lane) show comparison of gene expression in whole leaves (lanes 1 and 3) and in isolated peltate glandular trichomes (lanes 2 and 4) from basil lines EMX-1 (lanes 1 and 2; produces para-O-methylated phenylpropenes) and SW (lanes 3 and 4; produces non-para-O-methylated phenylpropenes). Lanes 5 to 7 (5 μg of total RNA per lane) show comparison of gene expression between different stages of leaf development from EMX-1 whole leaves 0.5 cm long (lane 5), 1 cm long (lane 6), and 3 cm long (lane 7). Lanes 8 to 12 (5 μg of total RNA per lane) show comparison of gene expression in different parts of EMX-1 leaves: lane 8, 1-cm leaf apical half; lane 9, 1-cm leaf basal half; lane 10, 3-cm leaf apical third; lane 11, 3-cm leaf middle third; lane 12, 3-cm leaf basal third. (B) Specific activities for EOMT (black bars) and CVOMT (white bars) enzymes. Lane numbers indicate tissues as described for (A). Bars indicate ±se.

Figure 5.

Comparison of Relative Specific Activities of Basil CVOMT1, EOMT1, and COMT1 with a Variety of Substrates. For each enzyme, the specific activity that is the highest is set at 100%; although EOMT has similar activity with chavicol as CVOMT, its activity with eugenol is fourfold higher.

Figure 6.

Stereo Views of the Three-Dimensional Structures of the Active Sites of Basil CVOMT (A), Basil EOMT (B), and C. breweri IEMT (C) as Determined by Molecular Modeling Based on the Crystal Structure of Alfalfa IOMT. The most efficient phenylpropene substrate for each enzyme is shown bound, as is S-adenosylhomocysteine (the cofactor product), which was cocrystalized with IOMT to produce the original three-dimensional structure.

Figure 7.

Substrate Specificity of Mutant Basil Phenylpropene OMTs. The relative specific activities of mutant basil CVOMT1 (CVOMT1 F260S) and EOMT1 (EOMT1 S261F) are compared with the specific activities of the corresponding native enzymes (CVOMT1 and EOMT1, respectively) using key substrates: chavicol (white bars), eugenol (black bars), t-isoeugenol (diagonally hatched bars), and caffeic acid (cross-hatched bars). For each enzyme, the specific activity that is the highest is set as 100%.