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.
2-Phenylethanol (2PE) is a prominent scent compound released from flowers of Damask roses (Rosa×damascena) and some hybrid roses (Rosa 'Hoh-Jun' and Rosa 'Yves Piaget'). 2PE is biosynthesized from l-phenylalanine (l-Phe) via the intermediate phenylacetaldehyde (PAld) by two key enzymes, aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (PAR). Here we describe substrate specificity and cofactor preference in addition to molecular characterization of rose-PAR and recombinant PAR from R.×damascena. The deduced amino acid sequence of the full-length cDNA encoded a protein exhibiting 77% and 75% identity with Solanum lycopersicum PAR1 and 2, respectively. The transcripts of PAR were higher in petals than calyxes and leaves and peaking at the unfurling stage 4. Recombinant PAR and rose-PAR catalyzed reduction of PAld to 2PE using NADPH as the preferred cofactor. Reductase activity of rose-PAR and recombinant PAR were higher for aromatic and aliphatic aldehydes than for keto-carbonyl groups. Both PARs showed that (S)-[4-(2)H] NADPH was preferentially used over the (R)-[4-(2)H] isomer to give [1-(2)H]-2PE from PAld, indicating that PAR can be classified as short-chain dehydrogenase reductase (SDR).
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