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首页> 外文期刊>Journal of Molecular Biology >Acetylation of the chemicals response regulator CheY by acetyl-CoA synthetase purified from Escherichia coli
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Acetylation of the chemicals response regulator CheY by acetyl-CoA synthetase purified from Escherichia coli

机译：从大肠杆菌中纯化的乙酰辅酶A合成酶将化学反应调节剂CheY乙酰化

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Acetylation of CheY, the excitatory response regulator of bacterial chemotaxis, by the enzyme acetyl-CoA synthetase (Acs) is involved in Escherichia coli chemotaxis, but its function is obscure. Here, we overproduced Acs from E. coli, purified it in quantities sufficient for biochemical work, and characterized both the enzyme and the CheY acetylation reaction that it catalyzes. Such characterization is essential for revealing the function of CheY acetylation in chemotaxis. The enzyme exhibited characteristics typical of prokaryotic Acs enzymes, and it could use either acetate or AcCoA as an acetyl donor for CheY acetylation. The Acs-catalyzed acetylation of CheY was reversible, an essential property for a regulatory process, and cooperative (Hill coefficient approximate to3). By Western blotting with specific anti-acetyl-lysine antibody we demonstrated that Acs undergoes autoacetylation, that CheY is acetylated to a small extent when isolated, and that the extent is elevated following in vitro acetylation. Exposing the intact protein to matrix-assisted laser desorption ionization time-of-flight mass spectrometry and electrospray mass spectrometry, we found that, in most cases, purified CheY is a mixture of species having zero to six acetyl groups per molecule, with nonacetylated CheY being the most abundant species. By proteolytic in-gel digestion of non-treated CheY followed by peptide fingerprinting, precursor ion scan, and tandem mass spectrometry, we found that the acetylation sites of CheY are clustered at the C terminus of the protein, with lysine residues 91, 92, 109, 119, 122 and 126 being the main acetylation sites. Following in vitro acetylation, the main change that seemed to occur was an incremental increase in the extent of acetylation of the same lysine residues. Thus, CheY is similar to many eukaryotic proteins involved in signaling, which undergo both phosphorylation and multiple acetylation, and in which the acetylation sites are restricted to a particular region. (C) 2004 Elsevier Ltd. All rights reserved.
机译：大肠杆菌趋化性涉及乙酰辅酶A合成酶（Acs）对细菌趋化性的兴奋反应调节剂CheY的乙酰化作用，但其功能尚不清楚。在这里，我们从大肠杆菌中过量生产了Acs，对其进行了足够的纯化以用于生化工作，并表征了其催化的酶和CheY乙酰化反应。此类表征对于揭示CheY乙酰化在趋化性中的功能至关重要。该酶具有原核Acs酶的典型特征，可以使用乙酸盐或AcCoA作为CheY乙酰化的乙酰基供体。 CheY的Acs催化乙酰化是可逆的，这是调节过程的基本特性，并且具有协同作用（希尔系数约为3）。通过用特异性抗乙酰赖氨酸抗体进行的蛋白质印迹，我们证明了Acs会发生自乙酰化，分离后CheY的乙酰化程度很小，并且在体外乙酰化后程度升高。将完整蛋白暴露于基质辅助激光解吸电离飞行时间质谱和电喷雾质谱中，我们发现，在大多数情况下，纯化的CheY是每个分子具有零至六个乙酰基的物质与未乙酰化的CheY的混合物是最丰富的物种。通过未经处理的CheY的蛋白水解凝胶电泳，然后进行肽指纹分析，前体离子扫描和串联质谱分析，我们发现CheY的乙酰化位点聚集在蛋白质的C末端，具有赖氨酸残基91、92， 109、119、122和126是主要的乙酰化位点。在体外乙酰化之后，似乎发生的主要变化是相同赖氨酸残基的乙酰化程度逐渐增加。因此，CheY与许多参与信号转导的真核蛋白质相似，它们会同时经历磷酸化和多重乙酰化作用，并且其中的乙酰化位点仅限于特定区域。 （C）2004 Elsevier Ltd.保留所有权利。

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