Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles.: Identification of the crucial role of a threonine residue for enzyme activity
Functional characterization of a melon alcohol acyl-transferase gene family involved in the biosynthesis of ester volatiles.: Identification of the crucial role of a threonine residue for enzyme activity
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DOI：
10.1007/s11103-005-8884-y
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发表时间：
2005-09-01
影响因子：
5.1
通讯作者：
Pech, JC
中科院分区：
文献类型：
--
作者：
El-Sharkawy, I;Manríquez, D;Pech, JC
挥发性酯是一类主要的水果香气成分，由醇酰基转移酶（AAT）合成。我们在这里证明，在夏伦泰斯甜瓜（Cucumis梅洛var. cantalupensis），AAT由至少四个成员的基因家族编码，其氨基酸同一性范围为84%（Cm-AAT 1/Cm-AAT 2）和58%（Cm-AAT 1/Cm-AAT 3）至仅22%（Cm-AAT 1/Cm-AAT 4）。所有编码的蛋白质，除了Cm-AAT 2，在酵母中表达时具有酶活性，并显示出不同的底物偏好。Cm-AAT 1蛋白产生宽范围的短链和长链酰基酯，但对形成E-2-己烯基乙酸酯和己酸己酯具有强烈的偏好。Cm-AAT 3也接受广泛的底物，但对生产乙酸苄酯具有非常强的偏好。Cm-AAT 4几乎专门用于形成乙酸酯，强烈偏好乙酸肉桂酯。定点诱变表明，Cm-AAT 2不能产生挥发性酯与268位丙氨酸残基的存在有关，而不是所有活性AAT蛋白中的苏氨酸。将Cm-AAT 2的268-A突变为268-T恢复了酶活性，而将268-T突变为268-A则消除了Cm-AAT 1的活性。这三种蛋白质的活性在果实成熟过程中均急剧增加。所有Cm-AAT基因的表达在成熟过程中被上调，并在反义ACC氧化酶甜瓜和用乙烯拮抗剂1-甲基环丙烯（1-MCP）处理的果实中被抑制，表明乙烯的正调控。在这项工作中提出的数据表明，AAT基因的多样性占甜瓜形成的酯的巨大多样性。
Volatile esters, a major class of compounds contributing to the aroma of many fruit, are synthesized by alcohol acyl-transferases (AAT). We demonstrate here that, in Charentais melon (Cucumis melo var. cantalupensis), AAT are encoded by a gene family of at least four members with amino acid identity ranging from 84% (Cm-AAT1/Cm-AAT2) and 58% (Cm-AAT1/Cm-AAT3) to only 22% (Cm-AAT1/Cm-AAT4). All encoded proteins, except Cm-AAT2, were enzymatically active upon expression in yeast and show differential substrate preferences. Cm-AAT1 protein produces a wide range of short and long-chain acyl esters but has strong preference for the formation of E-2-hexenyl acetate and hexyl hexanoate. Cm-AAT3 also accepts a wide range of substrates but with very strong preference for producing benzyl acetate. Cm-AAT4 is almost exclusively devoted to the formation of acetates, with strong preference for cinnamoyl acetate. Site directed mutagenesis demonstrated that the failure of Cm-AAT2 to produce volatile esters is related to the presence of a 268-alanine residue instead of threonine as in all active AAT proteins. Mutating 268-A into 268-T of Cm-AAT2 restored enzyme activity, while mutating 268-T into 268-A abolished activity of Cm-AAT1. Activities of all three proteins measured with the prefered substrates sharply increase during fruit ripening. The expression of all Cm-AAT genes is up-regulated during ripening and inhibited in antisense ACC oxidase melons and in fruit treated with the ethylene antagonist 1-methylcyclopropene (1-MCP), indicating a positive regulation by ethylene. The data presented in this work suggest that the multiplicity of AAT genes accounts for the great diversity of esters formed in melon.