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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
doi: 10.11833/j.issn.2095-0756.20220764
Identification of key amino acid residues controlling the activities of glycerol-3-phosphate acyltransferases in Arabidopsis thaliana and Brassica napus
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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AtGPAT9和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位同时存在磷酸化氨基酸残基对酰基转移酶活性有害，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GAPT9活性。图7表2参37 Abstract: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, W85 and H119 are prerequisite for the proper functioning of GPAT9, and L114, D230, N237, and A322 are beneficial to maintaining the activity of GPAT9. [Ch, 7 fig. 2 tab. 37 ref.] -
图 1 拟南芥AtGPAT9和油菜BnGPAT9氨基酸序列比对
Figure 1 Alignment of the amino acid sequences of A. thaliana AtGPAT9 and B. napus BnGPAT9
图 2 AtGPAT9和BnGPAT9三维结构预测
Figure 2 Prediction of three-dimensional structures of AtGPAT9 and BnGPAT9
图 3 不同单位点突变对AtGPAT9和BnGPAT9酶活性的影响
Figure 3 Effects of different single mutations on AtGPAT9 and BnGPAT9 activities
图 4 亮氨酸替换114位的苏氨酸(T114L)对BnGPAT9活性的影响
Figure 4 Effects of the substitution of threonine for leucine at residue 114 (T114L) on BnGPAT9 activity
图 5 多位点突变对AtGPAT9酶活性的影响
Figure 5 Effects of mutations at multiple sites on AtGPAT9 activity
图 6 含不同突变位点的AtGPAT9表达对不同生长期酵母菌株ZAFU1的细胞密度影响
Figure 6 Effects of expression of AtGPAT9 bearing different mutation sites on cell density of the yeast strain ZAFU1 in different periods of growth
图 7 含不同突变位点的AtGPAT9表达对平台生长期酵母菌株ZAFU1的TAG含量影响
Figure 7 Effects of expression of AtGPAT9 bearing different mutation sites on TAG content of the yeast strain ZAFU1 at the stationary phase
表 1 拟南芥AtGPAT9和油菜BnGPAT9定点突变所用的引物序列
Table 1. Sequences of the primers used for site-directed mutagenesis of A. thaliana AtGPAT9 and B. napus BnGPAT9
突变位点 引物序列 (5′→3′) 突变位点 引物序列 (5′→3′) BnGPAT9(R40S) AGCCTCGTGGCAAGCTCAGCCTGCGTGATTTGCTAGACAT AtGPAT9(N119H) TTTCATTGTTTATCCCTGTACACGCGTTGCTGAAAGGTCAAG BnGPAT9(W85Y) TCTACTTGTTTCCTTTATACTGCTGTGGTGTTGTTGTTAG AtGPAT9(D230N) TTGTAGCAAAAAAGTTAAGGAACCATGTCCAAGGAGCTGAC BnGPAT9(C87F) TTGTTTCCTTTATGGTGCTTTGGTGTTGTTGTTAGATACT AtGPAT9(A235T) TAAGGGACCATGTCCAAGGAACTGACAGTAATCCTCTTCTC BnGPAT9(I102F) TTCTCTTTCCCTTGAGGTGCTTCACTTTAGCTTTTGGATG AtGPAT9(S237N) ACCATGTCCAAGGAGCTGACAATAATCCTCTTCTCATATTTCC BnGPAT9(F109I) CATCACTTTAGCTTTTGGATGGATTATTTTCCTTTCAACG AtGPAT9(D230N/A235T) TTGTAGCAAAAAAGTTAAGGAACCATGTCCAAGGAACTGAC BnGPAT9(T114L) TGGTTTATTTTCCTTTCATTGTTTATCCCTGTACACTCTC AtGPAT9(A235T/ S237N) TAAGGGACCATGTCCAAGGAACTGACAATAATCCTCTTCTCATATTTC BnGPAT9(H119N) TTCAACGTTTATCCCTGTAAATTCTCTCCTGAAAGGTCAG AtGPAT9(D230N/A235T/S237N) TTGTAGCAAAAAAGTTAAGGAACCATGTCCAAGGAACTGAC BnGPAT9(N230D) GTAGCAAGAAAGTTAAGGGACCATGTTCAAGGAACTGACA AtGPAT9(G332A) CATAAGGCCCGGTGAAACAGCAATTGAATTTGCAGAGAGGG BnGPAT9(T235A) TAAGGAACCATGTTCAAGGAGCTGACAATAACCCTCTTCT AtGPAT9(L335H) GGTCAGAGACATGATATCTCATCGGGCGGGTCTCAAAAAGG BnGPAT9(N237S) CATGTTCAAGGAACTGACAGTAACCCTCTTCTTATATTTC AtGPAT9(P355S) TGAAGTATTCGAGACCAAGCTCCAAGCATAGTGAACGCAAG BnGPAT9(A322G) AAGGCCTGGTGAAACAGGAATTGAGTTTGCAGAGAGGGTC AtGPAT9(T10A) GTACGGCAGGGAGGCTCGTGGCTTCAAAATCCGAGCTTGAC AtGPAT9(S40R) ATGAACCTCGCGGCAAGCTCCGCCTGCGTGATTTGCTAGA AtGPAT9(S11A) CGGCAGGGAGGCTCGTGACTGCAAAATCCGAGCTTGACCTC AtGPAT9(Y85W) ATTTACTTATTCCCACTATGGTGCTTTGGGGTTGTTGTTAG AtGPAT9(S13A) GGAGGCTCGTGACTTCAAAAGCCGAGCTTGACCTCGATCAC AtGPAT9(F87C) CTTATTCCCACTATACTGCTGTGGGGTTGTTGTTAGATACT AtGPAT9(S28A) AACATCGAAGATTACCTTCCTGCTGGTTCTTCCATCAATGAAC AtGPAT9(F102I) TCCTCTTTCCCTTGAGGTGCATCACTTTAGCTTTTGGGTGG AtGPAT9(S30A) GAAGATTACCTTCCTTCTGGTGCTTCCATCAATGAACCTCGCG AtGPAT9(I109F) TCACTTTAGCTTTTGGGTGGTTTATTTTCCTTTCATTGTTT AtGPAT9(S31A) GATTACCTTCCTTCTGGTTCTGCCATCAATGAACCTCGCGGCA AtGPAT9(L114T) GGGTGGATTATTTTCCTTTCAACGTTTATCCCTGTAAATGCG 
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表 2 AtGPAT9和BnGPAT9中单和多位点氨基酸残基的定点突变
Table 2. Site-directed mutagenesis of amino acid residues at single and multiple sites in AtGPAT9 and BnGPAT9
单个氨基酸残基突变 AtGPAT9氨基酸残基突变组合 BnGPAT9 AtGPAT9 BnR40S** AtT10A AtF102I/S237N AtY85W/D230N BnW85Y AtS11A AtI109F/S237N AtY85W/A235T BnC87F** AtS13A AtD230N/A235T AtY85W/S237N** BnI102F** AtS28A AtD230N/S237N AtY85W/D230N/A235T BnF109I** AtS30A AtA235T/S237N AtY85W/D230N/S237N BnT114L*** AtS31A AtD230N/A235T/S237N AtY85W/A235T/S237N BnH119N AtS40R AtS237N/G322A AtY85W/D230N/A235T/S237N BnN230D*** AtY85W AtY85W/N119H*** AtS40R/Y85W/S237N** BnT235A** AtF87C AtY85W/L114T/N119H* AtN119H/D230N BnN237S* AtF102I AtY85W/N119H/S237N*** AtN119H/A235T BnA322G* AtI109F AtY85W/L114T/N119H/S237N*** AtN119H/S237N*** AtL114T AtY85W/N119H/D230N** AtN119H/D230N/A235T AtN119H* AtY85W/N119H/A235T** AtN119H/D230N/S237N** AtD230N AtN119H/A235T/S237N*** AtA235T AtN119H/D230N/A235T/S237N AtS237N AtG322A AtL335H AtP355S 说明：每种突变以物种的首字母缩写和突变前后的氨基酸残基缩写表示，如AtS40R/S237N代表AtGPAT9 40位由丝氨酸(S)变为精氨酸(R)，237位由丝氨酸(S)变为天冬酰胺(N)。*代表基因的异源表达能够恢复酵母双突变体ZAFU1的生长缺陷；*数目代表恢复能力的大小，数目越多，能力越强
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