Site-directed mutagenesis identified the key active site residues of alcohol acyltransferase PpAAT1 responsible for aroma biosynthesis in peach fruits.
Site-directed mutagenesis identified the key active site residues of alcohol acyltransferase PpAAT1 responsible for aroma biosynthesis in peach fruits.
复制标题
定点诱变鉴定了负责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
DOI：
10.1038/s41438-021-00461-x
复制
发表时间：
2021-02-01
影响因子：
8.7
通讯作者：
Zhang HX
中科院分区：
文献类型：
--
作者：
Song ZZ;Peng B;Gu ZX;Tang ML;Li B;Liang MX;Wang LM;Guo XT;Wang JP;Sha YF;Zhang HX
桃果实的香气主要由γ-癸内酯和酯类化合物的积累决定。先前的研究表明，这些香气化合物在桃果实中的生物合成是由醇酰基转移酶PpAAT 1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。共14个候选氨基酸残基可能参与内部酯化和9个候选氨基酸残基可能参与酯化的PpAAT 1进行了评估，通过定点突变。PpAAT 1及其定点突变蛋白的体外酶活性分析对不同氨基酸突变位点的PpAAT 1-SMs进行了比较，并对瞬时表达PpAAT 1和PpAAT 1-SMs的转基因烟草叶片和桃果实中γ-癸内酯的含量进行了分析，结果表明，在保守的HxxxD基序中的H165的定向突变导致PpAAT 1在内部酯化及其反应中的酶活性丧失，而关键氨基酸残基D376的突变导致PpAAT 1的γ-癸内酯生物合成活性完全丧失。其他9个和7个氨基酸残基的突变也显着影响PpAAT 1的酶活性的内部酯化和酯化反应，分别。研究结果为PpAAT 1催化桃果实中γ-癸内酯和酯类化合物的机械合成提供了生化基础，可用于指导桃新品种的分子育种。
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.
登录
查看更多内容
DOI：
10.1111/nph.14970
发表时间：
2018-04
期刊：
The New phytologist
影响因子：
--
作者：
de Souza WR;Martins PK;Freeman J;Pellny TK;Michaelson LV;Sampaio BL;Vinecky F;Ribeiro AP;da Cunha BADB;Kobayashi AK;de Oliveira PA;Campanha RB;Pacheco TF;Martarello DCI;Marchiosi R;Ferrarese-Filho O;Dos Santos WD;Tramontina R;Squina FM;Centeno DC;Gaspar M;Braga MR;Tiné MAS;Ralph J;Mitchell RAC;Molinari HBC
通讯作者：
Molinari HBC
影响因子：
9.4
作者：
Ilc, Tina;Halter, David;Navrot, Nicolas
通讯作者：
Navrot, Nicolas
影响因子：
27.5
作者：
Goulet, Charles;Kamiyoshihara, Yusuke;Klee, Harry J.
通讯作者：
Klee, Harry J.
影响因子：
11.6
作者：
Kumar, Roshan;Lee, Soon Goo;Bisht, Naveen C.
通讯作者：
Bisht, Naveen C.
影响因子：
4.4
作者：
JORGENSEN, WL;CHANDRASEKHAR, J;KLEIN, ML
通讯作者：
KLEIN, ML