Biochemical Characterization of the Lysine Acetylation of Tyrosyl-tRNA Synthetase in Escherichia coli.
Biochemical Characterization of the Lysine Acetylation of Tyrosyl-tRNA Synthetase in Escherichia coli.
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DOI：
10.1002/cbic.201700343
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发表时间：
2017-10-05
期刊：
影响因子：
--
通讯作者：
Fan C
中科院分区：
文献类型：
--
作者：
Venkat S;Gregory C;Gan Q;Fan C
氨酰-tRNA合成酶（aminoacyl-tRNA synthetases，aaRSs）在蛋白质合成中起重要作用。作为阿尔斯家族的一员，大肠杆菌中的酪氨酰-tRNA合成酶（TyrRS）在蛋白质组学研究中已显示在多个赖氨酸残基处被乙酰化。然而，这些推定的乙酰化目标尚未进行生化表征。在这项研究中，我们应用了遗传密码扩展策略，将Nε-乙酰基-L-赖氨酸定点掺入TyrRS的选定位置，用于体外表征。酶活性测定表明，K85、K235和K238处的乙酰化会损害酶活性。体外脱乙酰实验表明，TyrRS中大部分乙酰化赖氨酸残基对大肠杆菌敏感。coli脱乙酰酶CobB而不是YcgC。体外乙酰化实验表明，大肠杆菌Gcn 5相关的N-乙酰转移酶家族的25个成员在大肠杆菌中表达。包括YfiQ在内的大肠杆菌不能有效地乙酰化TyrRS，而TyrRS只能被乙酰辅酶A或乙酰磷酸（AcP）化学乙酰化。我们的体外表征实验表明赖氨酸乙酰化可能是调节阿尔斯酶活性的可能机制，从而影响翻译。可逆赖氨酸乙酰化：位点特异性乙酰化的大肠杆菌酪氨酰-tRNA合成酶的变异体产生的遗传密码扩展策略。特定赖氨酸残基的乙酰化消除了氨酰化活性。赖氨酸残基在体外可被乙酰磷酸（AcP）化学乙酰化，并可被CobB脱乙酰酶脱乙酰化。
Aminoacyl-tRNA synthetases (aaRSs) play essential roles in protein synthesis. As a member of the aaRS family, the tyrosyl-tRNA synthetase (TyrRS) in Escherichia coli has been shown in proteomic studies to be acetylated at multiple lysine residues. However, these putative acetylation targets have not yet been biochemically characterized. In this study, we applied a genetic-code-expansion strategy to site-specifically incorporate Nε-acetyl-L-lysine into selected positions of TyrRS for in vitro characterization. Enzyme assays demonstrated that acetylation at K85, K235, and K238 could impair the enzyme activity. In vitro deacetylation experiments showed that most acetylated lysine residues in TyrRS were sensitive to the E. coli deacetylase CobB but not YcgC. In vitro acetylation assays indicated that 25 members of the Gcn5-related N-acetyltransferase family in E. coli, including YfiQ, could not acetylate TyrRS efficiently, whereas TyrRS could be acetylated chemically by acetyl-CoA or acetyl-phosphate (AcP) only. Our in vitro characterization experiments indicated that lysine acetylation could be a possible mechanism for modulating aaRS enzyme activities, thus affecting translation. Reversible lysine acetylation: Site-specifically acetylated Escherichia coli tyrosyl-tRNA synthetase variants were generated by a genetic code expansion strategy. The acetylation of specific lysine residues abolished the aminoacylation activity. The lysine residues could be chemically acetylated by acetyl-phosphate (AcP) and deacetylated by CobB deacetylase in vitro.
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影响因子：
14.9
作者：
Fan C;Xiong H;Reynolds NM;Söll D
通讯作者：
Söll D
影响因子：
14.8
作者：
Guo, Min;Schimmel, Paul
通讯作者：
Schimmel, Paul
影响因子：
5.6
作者：
BRICK, P;BLOW, DM
通讯作者：
BLOW, DM
DOI：
10.1126/stke.2452004pe42
发表时间：
2004-08-03
期刊：
Science's STKE : signal transduction knowledge environment
影响因子：
--
作者：
Cohen, Todd;Yao, Tso-Pang
通讯作者：
Yao, Tso-Pang
影响因子：
3.4
作者：
AbouElfetouh, Alaa;Kuhn, Misty L.;Hu, Linda I.;Scholle, Michael D.;Sorensen, Dylan J.;Sahu, Alexandria K.;Becher, Doerte;Antelmann, Haike;Mrksich, Milan;Anderson, Wayne F.;Gibson, Bradford W.;Schilling, Birgit;Wolfe, Alan J.
通讯作者：
Wolfe, Alan J.