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Article|01 Feb 2021|OPEN
Site-directed mutagenesis identified the key active site residues of alcohol acyltransferase PpAAT1 responsible for aroma biosynthesis in peach fruits
Zhi-Zhong Song1,2, Bin Peng2,1,, Zi-Xia Gu3, Mei-Ling Tang2,4, Bei Li1,2, Mei-Xia Liang1,2, Li-Min Wang1,2, Xiao-Tong Guo1,2, Jian-Ping Wang4, Yu-Fen Sha4 & Hong-Xia Zhang1,2,
1The Engineering Research Institute of Agriculture and Forestry, Ludong University, 186 Hongqizhong Road, Yantai 264025, China
2Key Laboratory of Molecular Module-Based Breeding of High Yield and Abiotic Resistant Plants in Universities of Shandong (Ludong University), 186 Hongqizhong Road, Yantai 264025, China
3Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, 1 Qianhuhoucun, Nanjing 210014, China
4Yantai Academy of Agricultural Science, 26 Gangcheng West Street, Yantai 265500, China

Horticulture Research 8,
Article number: 32 (2021)
doi: 10.1038/hortres.2021.32
Views: 235

Received: 18 Jun 2020
Revised: 24 Oct 2020
Accepted: 13 Nov 2020
Published online: 01 Feb 2021

Abstract

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.