The aroma of peach fruit is predominantly determined by the accumulation of γ-decalactone and ester compounds. A previous study showed that the biosynthesis of these aroma compounds in peach fruit is catalyzed by PpAAT1, an alcohol acyltransferase. In this work, we investigated the key active site residues responsible for γ-decalactone and ester biosynthesis. A total of 14 candidate amino acid residues possibly involved in internal esterification and 9 candidate amino acid residues possibly involved in esterification of PpAAT1 were assessed via site-directed mutagenesis. Analyses of the in vitro enzyme activities of PpAAT1 and its site-directed mutant proteins (PpAAT1-SMs) with different amino acid residue mutations as well as the contents of γ-decalactone in transgenic tobacco leaves and peach fruits transiently expressing PpAAT1 and PpAAT1-SMs revealed that site-directed mutation of H165 in the conserved HxxxD motif led to lost enzymatic activity of PpAAT1 in both internal esterification and its reactions, whereas mutation of the key amino acid residue D376 led to the total loss of γ-decalactone biosynthesis activity of PpAAT1. Mutations of 9 and 7 other amino acid residues also dramatically affected the enzymatic activity of PpAAT1 in the internal esterification and esterification reactions, respectively. Our findings provide a biochemical foundation for the mechanical biosynthesis of γ-decalactone and ester compounds catalyzed by PpAAT1 in peach fruits, which could be used to guide the molecular breeding of new peach species with more favorable aromas for consumers.

中文翻译：

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定点诱变鉴定了桃果实香气生物合成的醇酰基转移酶PpAAT1的关键活性位点残基

桃果实的香气主要由γ-癸内酯和酯类化合物的积累决定。先前的一项研究表明，桃果实中这些香气化合物的生物合成是由醇酰基转移酶 PpAAT1 催化的。在这项工作中，我们研究了负责 γ-癸内酯和酯生物合成的关键活性位点残基。通过定点诱变评估了总共14个可能参与内部酯化的候选氨基酸残基和9个可能参与PpAAT1酯化的候选氨基酸残基。不同氨基酸残基突变的PpAAT1及其定点突变蛋白(PpAAT1-SMs)的体外酶活性分析以及瞬时表达PpAAT1和PpAAT1-SMs的转基因烟叶和桃果实中γ-癸内酯的含量分析揭示了在保守的 HxxxD 基序中 H165 的定点突变导致 PpAAT1 在内部酯化及其反应中失去酶活性，而关键氨基酸残基 D376 的突变导致完全丧失 PpAAT1 的 γ-癸内酯生物合成活性. 9 个和 7 个其他氨基酸残基的突变也分别显着影响了 PpAAT1 在内部酯化和酯化反应中的酶活性。