title: Benzenoid biosynthesis in the flowers of Eriobotrya japonica: molecular cloning and functional characterization of p-methoxybenzoic acid carboxyl methyltransferase.
abstract: p -Methoxybenzoic acid carboxyl methyltransferase (MBMT) was isolated from loquat flowers. MBMT displayed high similarity to jasmonic acid carboxyl methyltransferases, but exhibited high catalytic activity to form methyl p -methoxybenzoate from p -methoxybenzoic acid. Volatile benzenoids impart the characteristic fragrance of loquat (Eriobotrya japonica) flowers. Here, we report that loquat produces methyl p-methoxybenzoate, along with other benzenoids, as the flowers bloom. Although the adaxial side of flower petals is covered with hairy trichomes, the trichomes are not the site of volatile benzenoid formation. Here we identified four carboxyl methyltransferase (EjMT1 to EjMT4) genes from loquat and functionally characterized EjMT1 which we found to encode a p-methoxybenzoic acid carboxyl methyltransferase (MBMT); an enzyme capable of converting p-methoxybenzoic acid to methyl p-methoxybenzoate via methylation of the carboxyl group. We found that transcript levels of MBMT continually increased throughout the flower development with peak expression occurring in fully opened flowers. Recombinant MBMT protein expressed in Escherichia coli showed the highest substrate preference toward p-methoxybenzoic acid with an apparent K m value of 137.3 µM. In contrast to benzoic acid carboxyl methyltransferase (BAMT) and benzoic acid/salicylic acid carboxyl methyltransferase, MBMT also displayed activity towards both benzoic acid and jasmonic acid. Phylogenetic analysis revealed that loquat MBMT forms a monophyletic group with jasmonic acid carboxyl methyltransferases (JMTs) from other plant species. Our results suggest that plant enzymes with same BAMT activity have evolved independently.
authors.affiliation: Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, 753-8515, Japan. takaori@yamaguchi-u.ac.jp.
authors.forename: Takao
authors.initials: T
authors.lastname: Koeduka
authors.affiliation: Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, 753-8515, Japan.
authors.forename: Mami
authors.initials: M
authors.lastname: Kajiyama
authors.affiliation: Department of Research and Development, Kazusa DNA Research Institute, Chiba, 292-0818, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan.
authors.forename: Takumi
authors.initials: T
authors.lastname: Furuta
authors.affiliation: Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan.
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Tsuge
authors.affiliation: Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, 753-8515, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Matsui
mesh_terms: D000595:Amino Acid Sequence; D031983:Eriobotrya / Q000201:enzymology* / Q000235:genetics; D035264:Flowers / Q000201:enzymology; D062425:Hydroxybenzoate Ethers / Q000378:metabolism*; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D018515:Plant Leaves / Q000201:enzymology; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017422:Sequence Analysis, DNA
chemical_list: D062425:Hydroxybenzoate Ethers; D010940:Plant Proteins; D008780:Methyltransferases
keywords: Carboxyl methyltransferase; Loquat; SABATH; Volatile benzenoids