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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 theSABATHfamily 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.
INTRODUCTION
Methylcinnamate and methyl-p-coumarate play important roles in plant-insect interactions and are widely distributed throughout the plant kingdom. They have been identified in both aerial and underground tissues from monocots and dicots as well as from ferns (Konda and Kawazu, 1979; Schaefers and Herrmann, 1982; Williams and Whitten, 1983; Hooper et al., 1984; Bandara et al., 1988; Cambie et al., 1990; Garcia et al., 1990; Seifert and Unger, 1994; Hiraga et al., 1996; Daayf et al., 1997a; Odell et al., 1999; Wu et al., 1999; Dias et al., 2003; Bruni et al., 2004; Azah et al., 2005; bin Jantan et al., 2005). Methylcinnamate, a known component of the floral scent from various orchid species, acts as a preferred attractant for euglossine bees and is electrophysiologically active toward excised bee antennae (Dodson et al., 1969; Ackerman, 1989; Schiestl and Roubik, 2003; Eltz and Lunau, 2005). Methylcinnamate production, however, is not strictly associated with flowers and has been found to accumulate to high levels in other tissues, including the leaves of sweet basil (Ocimum basilicum). In contrast...