Identification of specific residues involved in substrate discrimination in two plant O-methyltransferases.
Among the large number of plant O-methyltransferases that are involved in secondary metabolism, only a few have been enzymatically characterized, and little information is available on the structure of their substrate binding site and the mechanism which determines their substrate specificity and methylation regiospecificity. We have previously reported the isolation of two O-methyltransferases, S-adenosyl-l-methionine:(iso)eugenol O-methyltransferase (IEMT) and S-adenosyl-l-methionine:caffeic acid O-methyltransferase (COMT) from Clarkia breweri, an annual plant from California. While IEMT and COMT (which methylate eugenol/isoeugenol and caffeic acid/5-hydroxyferulic acid, respectively) share 83% identity at the amino acid level, they have distinct substrate specificity and methylation regiospecificity. We report here that seven amino acids play a critical role in discriminating between eugenol/isoeugenol and caffeic acid/5-hydroxyferulic acid. When these amino acids in IEMT were replaced by the corresponding residues of COMT, the hybrid protein showed activity only with caffeic acid/5-hydroxyferulic acid. Conversely, when these amino acids in COMT were replaced by corresponding IEMT residues, the hybrid protein had activity only with eugenol/isoeugenol. These results provide strong evidence that O-methyltransferase substrate preference could be determined by a few amino acid residues and that new OMTs with different substrate specificity could begin to evolve from an existing OMT by mutation of a few amino acids. Phylogenetic analysis confirms that C. breweri IEMT evolved recently from COMT.
Biology Department, University of Michigan, Ann Arbor, Michigan, 48109-1048, USA.
J
J
Wang
E
E
Pichersky
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D007700:Kinetics; D019684:Magnoliopsida / Q000201:enzymology* / Q000235:genetics; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D008780:Methyltransferases; C106569:eugenol O-methyltransferase; C019608:caffeate O-methyltransferase