title: Functional characterization of aromatic amino acid aminotransferase involved in 2-phenylethanol biosynthesis in isolated rose petal protoplasts.
abstract: In rose flowers, 2-phenylethanol (2PE) is biosynthesized from l-phenylalanine (l-Phe) via phenylacetaldehyde (PAld) by the actions of two enzymes, pyridoxal-5'-phosphate (PLP)-dependent aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). We here report that Rosa 'Yves Piaget' aromatic amino acid aminotransferase produced phenylpyruvic acid (PPA) from l-Phe in isolated petal protoplasts. We have cloned three full length cDNAs (RyAAAT1-3) of aromatic amino acid aminotransferase families based on rose EST database and homology regions. The RyAAATs enzymes were heterogeneously expressed in Escherichia coli and characterized biochemically. The recombinant RyAAAT3 showed the highest activity toward l-Phe in comparison with l-tryptophan, l-tyrosine, d-Phe, glycine, and l-alanine, and showed 9.7-fold higher activity with l-Phe rather than PPA as a substrate. RyAAAT3 had an optimal activity at pH 9 and at 45-55°C with α-ketoglutaric acid, and was found to be a PLP dependent enzyme based on the inhibition test using Carbidopa, an inhibitor of PLP-dependent enzymes. The transcript of RyAAAT3 was expressed in flowers as well as other organs of R. 'Yves Piaget'. RNAi suppression of RyAAAT3 decreased 2PE production, revealing the involvement of RyAAAT3 in 2PE biosynthesis in rose protoplasts and indicating that rose protoplasts have potentially two different 2PE biosynthetic pathways, the AADC route and the new route via PPA from l-Phe.
authors.affiliation: Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hirata
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Ohnishi
authors.forename: Haruka
authors.initials: H
authors.lastname: Ishida
authors.forename: Kensuke
authors.initials: K
authors.lastname: Tomida
authors.forename: Miwa
authors.initials: M
authors.lastname: Sakai
authors.forename: Masakazu
authors.initials: M
authors.lastname: Hara
authors.forename: Naoharu
authors.initials: N
authors.lastname: Watanabe
mesh_terms: D053898:Biosynthetic Pathways; D035264:Flowers / Q000201:enzymology / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D010626:Phenylethyl Alcohol / Q000378:metabolism*; D010937:Plant Growth Regulators / Q000378:metabolism; D030821:Plants, Genetically Modified; D011523:Protoplasts / Q000201:enzymology* / Q000378:metabolism; D027826:Rosa / Q000201:enzymology* / Q000378:metabolism; D000637:Transaminases / Q000378:metabolism*
chemical_list: D010937:Plant Growth Regulators; D000637:Transaminases; C019729:aromatic amino acid aminotransferase; D010626:Phenylethyl Alcohol