|Year of Publication:
|J. Kapteyn, Qualley, A. V., Xie, Z., Fridman, E., Dudareva, N., Gang, D. R.
|Amino Acid Sequence, Blotting: Southern, Cinnamates, Evolution: Molecular, Expressed Sequence Tags, Gas Chromatography-Mass Spectrometry, Gene Expression Profiling, Kinetics, Methylation, Methyltransferases, Models: Molecular, Molecular Sequence Data, Molecular Structure, Ocimum basilicum, Phylogeny, Plant Proteins, Protein Isoforms, Proteomics, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology: Amino Acid, Substrate Specificity
Methylcinnamate, which is widely distributed throughout the plant kingdom, is a significant component of many floral scents and an important signaling molecule between plants and insects. Comparison of an EST database obtained from the glandular trichomes of a basil (Ocimum basilicum) variety that produces high levels of methylcinnamate (line MC) with other varieties producing little or no methylcinnamate identified several very closely related genes belonging to the SABATH family of carboxyl methyltransferases that are highly and almost exclusively expressed in line MC. Biochemical characterization of the corresponding recombinant proteins showed that cinnamate and p-coumarate are their best substrates for methylation, thus designating these enzymes as cinnamate/p-coumarate carboxyl methyltransferases (CCMTs). Gene expression, enzyme activity, protein profiling, and metabolite content analyses demonstrated that CCMTs are responsible for the formation of methylcinnamate in sweet basil. A phylogenetic analysis of the entire SABATH family placed these CCMTs into a clade that includes indole-3-acetic acid carboxyl methyltransferases and a large number of uncharacterized carboxyl methyltransferase-like proteins from monocots and lower plants. Structural modeling and ligand docking suggested active site residues that appear to contribute to the substrate preference of CCMTs relative to other members of the SABATH family. Site-directed mutagenesis of specific residues confirmed these findings.