Department of Bacteriology, University of Wisconsin, Madison, Wisconsin, USA.
mBio. 2011 Oct 18;2(5). doi: 10.1128/mBio.00216-11. Print 2011.
In the bacterium Salmonella enterica, the CobB sirtuin protein deacetylase and the Gcn5-related N(ε)-acetyltransferase (GNAT) Pat control carbon utilization and metabolic flux via N(ε)-lysine acetylation/deacetylation of metabolic enzymes. To date, the S. enterica Pat (SePat) acetyltransferase has not been biochemically characterized. Here we report the kinetic and thermodynamic characterization of the SePat enzyme using two of its substrates, acetyl coenzyme A (Ac-CoA) synthetase (Acs; AMP forming, EC 6.2.1.1) and Ac-CoA. The data showed typical Michaelis-Menten kinetic behavior when Ac-CoA was held at a saturating concentration while Acs was varied, and a sigmoidal kinetic behavior was observed when Acs was saturating and the Ac-CoA concentration was varied. The observation of sigmoidal kinetics and positive cooperativity for Ac-CoA is an unusual feature of GNATs. Results of isothermal titration calorimetry (ITC) experiments showed that binding of Ac-CoA to wild-type SePat produced a biphasic curve having thermodynamic properties consistent with two distinct sites. Biphasicity was not observed in ITC experiments that analyzed the binding of Ac-CoA to a C-terminal construct of SePat encompassing the predicted core acetyltransferase domain. Subsequent analytical gel filtration chromatography studies showed that in the presence of Ac-CoA, SePat oligomerized to a tetrameric form, whereas in the absence of Ac-CoA, SePat behaved as a monomer. The positive modulation of SePat activity by Ac-CoA, a product of the Acs enzyme that also serves as a substrate for SePat-dependent acetylation, is likely a layer of metabolic control. IMPORTANCE For decades, N(ε)-lysine acetylation has been a well-studied mode of regulation of diverse proteins involved in almost all aspects of eukaryotic physiology. Until recently, N(ε)-lysine acetylation was not considered a widespread phenomenon in bacteria. Recent studies have indicated that N(ε)-lysine acetylation and its impact on cellular metabolism may be just as diverse in bacteria as they are in eukaryotes. The S. enterica Pat enzyme, specifically, has recently been implicated in the modulation of many metabolic enzymes. Understanding the molecular mechanisms of how this enzyme controls the activity of diverse enzymes by N(ε)-lysine acetylation will advance our understanding of how the prokaryotic cell responds to its changing environment in order to meet its metabolic needs.
在细菌沙门氏菌中，CobB 组蛋白去乙酰化酶和 Gcn5 相关的 N(ε)-乙酰转移酶（GNAT）Pat 通过代谢酶的 N(ε)-赖氨酸乙酰化/去乙酰化来控制碳利用和代谢通量。迄今为止，尚未对沙门氏菌的 Pat（SePat）乙酰转移酶进行生化表征。在这里，我们使用其两种底物乙酰辅酶 A（Ac-CoA）合成酶（Acs；AMP 形成，EC 6.2.1.1）和 Ac-CoA 来报告 SePat 酶的动力学和热力学特征。当 Ac-CoA 保持在饱和浓度而 Acs 变化时，数据显示出典型的米氏动力学行为，而当 Acs 饱和而 Ac-CoA 浓度变化时，观察到 Sigmoidal 动力学行为。观察到 Ac-CoA 的 Sigmoidal 动力学和正协同作用是 GNAT 的一个不寻常特征。等温滴定量热法（ITC）实验的结果表明，Ac-CoA 与野生型 SePat 的结合产生了具有与两个不同位点一致的热力学性质的双相曲线。在分析 Ac-CoA 与包含预测核心乙酰转移酶结构域的 SePat 末端结构域的结合的 ITC 实验中，未观察到双相性。随后的分析凝胶过滤色谱研究表明，在 Ac-CoA 的存在下，SePat 聚合为四聚体形式，而在没有 Ac-CoA 的情况下，SePat 表现为单体。Acs 酶的产物 Ac-CoA 对 SePat 活性的正向调节，也是一种代谢控制的方式。重要性几十年来，N(ε)-赖氨酸乙酰化一直是调节参与真核生理几乎所有方面的多种蛋白质的一种研究良好的调节模式。直到最近，N(ε)-赖氨酸乙酰化才被认为不是细菌中广泛存在的现象。最近的研究表明，N(ε)-赖氨酸乙酰化及其对细胞代谢的影响在细菌中可能与在真核生物中一样多样化。沙门氏菌 Pat 酶最近特别被牵连到许多代谢酶的调节中。了解该酶通过 N(ε)-赖氨酸乙酰化控制多种酶活性的分子机制，将有助于我们了解原核细胞如何响应不断变化的环境以满足其代谢需求。
