拟南芥和油菜3-磷酸甘油酰基转移酶的关键活性位点鉴定  

Identification of key amino acid residues controlling the activities of glycerol-3-phosphate acyltransferases in Arabidopsis thaliana and Brassica napus

作　　者：林怡馨 陈丹丹 刘宏波[1] 柯星星 郑月萍 郑志富[1,2] LIN Yixin;CHEN Dandan;LIU Hongbo;KE Xingxing;ZHENG Yueping;ZHENG Zhifu(College of Advanced Agricultural Sciences,Zhejiang A&F University,Hangzhou 311300,Zhejiang,China;Institute for Oilseed Crop Germplasm Innovation and Utilization,Zhejiang A&F University,Hangzhou 311300,Zhejiang,China)

机构地区：[1]浙江农林大学现代农学院,浙江杭州311300 [2]浙江农林大学油料作物种质创新与利用研究所,浙江杭州311300

出　　处：《浙江农林大学学报》2023年第4期695-706,共12页Journal of Zhejiang A&F University

基　　金：国家自然科学基金青年基金资助项目(32100209);国家自然科学基金面上资助项目(31871660,32071929);浙江省自然科学基金青年基金资助项目(Q21C020003)。

摘　　要：【目的】3-磷酸甘油酰基转移酶(GPAT)催化三酰甘油(TAG)生物合成途径中的第1步酰化反应,TAG合成能力是油料作物的关键性状,但亦与人类肥胖症密切相关,了解GPAT结构与功能的内在关系对于这一性状的遗传或化学遗传调控至关重要。本研究旨在鉴定调控植物GPAT活性的关键氨基酸位点。【方法】运用定点突变技术构建了58个GPAT9突变基因,结合GPAT特异的酵母遗传互补法,剖析单一和多个氨基酸位点改变对GPAT9酶活性的影响。【结果】通过对拟南芥Arabidopsis thaliana AtGPAT9和油菜Brassica napus BnGPAT9的19个氨基酸残基进行分析发现:AtGPAT9的N端6个磷酸化位点的单独突变(T10A、S11A、S13A、S28A、S30A和S31A)不能增强AtGPAT9在酵母异源表达时的活性。相反,其他6个位于酰基转移酶保守结构域外的氨基酸残基(85、114、119、230、237、322位)的改变能够显著影响GPAT9酶活性。发现这些氨基酸残基之间存在交互作用,例如,3个位点同时突变(Y85W/N119H/S237N)能使AtGPAT9活性大幅上升,加速酵母的生长并促进TAG的合成,表达这一突变酶的酵母中的TAG含量比表达野生型BnGPAT9的增加了45.7%。更值得注意的是,在114和237位磷酸化氨基酸残基对酰基转移酶活性产生负面效应,暗示植物GPAT9活性可能受磷酸化和非磷酸化机制调节。【结论】本研究获得了6个未经报道的关键GPAT酶活性调控位点,其中W85和H119是GPAT9正常功能所必需的,而L114、D230、N237和A322有利于维持GPAT9活性。[Objective]Glycerol-3-phosphate acyltransferase(GPAT)catalyzes the first acylation reaction in the triacylglycerol(TAG)biosynthetic pathway.Understanding the structure-function relationship of GPAT is important for genetic or chemogenetic manipulation of the TAG biosynthetic capacity as a key trait in oilseed crops but associated with human obesity disease.This study aimed to identify key amino acid residues controlling the activity of GPAT9 in plants.[Method]Site-directed mutagenesis was employed to construct 58 GPAT9 mutant genes,and yeast genetic complementation specific for GPAT enzyme was utilized to dissect the effects of alterations of single and multiple amino acid residues on the activities of GPAT9 from Arabidopsis thaliana and Brassica napus.[Result]By analyzing 19 amino acid residues of AtGPAT9 and BnGPAT9,it was found that single mutations(T10A,S11A,S13A,S28A,S30A,S31A)at the N-terminus of AtGPAT9 could not enhance its activity when the respective variants were expressed heterologously.In contrast,the alteration of the other six amino acid residues at positions 85,114,119,230,237,and 322,respectively,which are located outside the conserved domains of acyltransferases significantly affected GPAT9 enzymatic activity.Additionally,mutual interactions were evident between these amino acids.For instance,the simultaneous mutation of the three residues(Y85W,N119H,S237N)greatly increased the activity of AtGPAT9,as exemplified by the findings that the expression of the corresponding mutant enzyme could accelerate the growth of yeast cells and enhanced the synthesis of TAG by 45.7%,compared with that of yeast cells expressing BnGPAT9.Noticeably,the presence of potentially phosphorylated amino acids at positions 114 and 237 was detrimental to acyltransferase activity,implying that plant GPAT9 activity may be regulated through protein phosphorylation and non-phosphorylation.[Conclusion]This study describes six previously unreported amino acid residues key to the regulation of GPAT enzymatic activity.Among them,W8

关 键 词：拟南芥 油菜 3-磷酸甘油酰基转移酶 结构与功能关系 定点突变 酵母遗传互补 

分 类 号：Q946.5[生物学—植物学]