Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan.
J Plant Physiol. 2012 Mar 15;169(5):444-51. doi: 10.1016/j.jplph.2011.12.005. Epub 2012 Jan 9.
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.
在玫瑰花朵中，2-苯乙醇（2PE）是通过两种酶的作用，从 L-苯丙氨酸（L-Phe）生物合成而来的苯乙醛（PAld），这两种酶分别是吡哆醛-5'-磷酸（PLP）依赖性芳香族氨基酸脱羧酶（AADC）和苯乙醛还原酶（PAR）。我们在这里报告称，Rosa 'Yves Piaget' 芳香族氨基酸氨基转移酶能够在分离的花瓣原生质体中从 L-Phe 产生苯丙酮酸（PPA）。我们基于玫瑰 EST 数据库和同源区域，克隆了三个全长 cDNA（RyAAAT1-3）的芳香族氨基酸氨基转移酶家族。RyAAAT 酶在大肠杆菌中异源表达，并进行了生化特性分析。与 L-色氨酸、L-酪氨酸、D-Phe、甘氨酸和 L-丙氨酸相比，重组 RyAAAT3 对 L-Phe 的活性最高，而与 PPA 相比，其对 L-Phe 的活性高 9.7 倍。RyAAAT3 在 pH9 下、α-酮戊二酸存在时，在 45-55°C 下活性最高，根据对依赖于 PLP 的酶的抑制剂 Carbidopa 的抑制试验，发现它是一种依赖于 PLP 的酶。在 R. 'Yves Piaget' 的花和其他器官中均表达了 RyAAAT3 的转录本。RyAAAT3 的 RNAi 抑制降低了 2PE 的产生，表明 RyAAAT3 参与了玫瑰原生质体中 2PE 的生物合成，并表明玫瑰原生质体可能具有两条不同的 2PE 生物合成途径，即 AADC 途径和通过 L-Phe 生成 PPA 的新途径。
