Structure-function analyses on AcuB encoded by the Bacillus subtilis acuABC-operon involved in regulation of acetyl-CoA synthetase by post-translational lysine acetylation

细菌细胞必须科普快速变化的环境条件，包括营养物质可用性的改变。一种感知细胞代谢状态的方法是通过翻译后赖氨酸乙酰化来调节蛋白质功能。在细菌中，赖氨酸乙酰化由赖氨酸乙酰转移酶（KAT）酶促催化，所述KAT使用乙酰辅酶A作为供体分子用于赖氨酸侧链的乙酰化。乙酰化的赖氨酸侧链可以通过脱乙酰酶脱乙酰化，脱乙酰酶分为两组，即经典的Zn 2+依赖性脱乙酰酶（KDAC）和NAD+依赖性沉默调节蛋白。除了作为乙酰化的供体分子，乙酰辅酶A是一种中心代谢物。乙酰辅酶A可以通过AMP形成乙酰辅酶A合成酶（Acs）的活性产生。该反应分为两个半反应。在第一半反应，即腺苷酸化反应中，乙酸盐被腺苷酸化激活，产生乙酰-AMP。随后，在第二半反应中，乙酰基-AMP转化为硫酯乙酰基-CoA（硫酯形成）。在革兰氏阳性细菌物种枯草芽孢杆菌中，乙酰辅酶A合成酶阿克萨在acuABC操纵子的上游编码并被转录。acuABC操纵子最初被鉴定为参与丁二醇和乙偶姻催化。acuABC操纵子的基因产物的功能在机制上仅部分了解。基因acuA编码GNAT型KAT，以在C-末端赖氨酸侧链处乙酰化阿克萨，即K549，从而抑制阿克萨活性。AcuC构成经典的Zn 2+依赖性脱乙酰酶。已知AcuC能够脱乙酰化，从而（再）活化阿克萨。AcuA介导的K549-乙酰化作用导致AcsA活性失活的确切分子机制仅知之甚少。AcuB在AcuA、AcuC和阿克萨相互作用中的作用完全不清楚。有趣的是，其他细菌物种，如金黄色葡萄球菌，也有类似的基因组织。然而，在S.在金黄色葡萄球菌中，操纵子仅编码AcuA和AcuC，但缺失acuB基因。这表明AcuB可能对某些细菌菌株具有特异性。我们推测，AcuB，这是完全功能不明的，可能是一个重要的调节器，这种相互作用，即感知代谢状态，调整AcuA和/或AcuC活动，以调节阿克萨的活动。在B。通过与AcuC和/或AcuA以及与诸如NAD+、AMP、ATP或CoA/乙酰辅酶A的分子结合而与枯草杆菌AcuB结合，可能是使脱乙酰酶AcuC和/或乙酰转移酶AcuA的活性适应于能够协调阿克萨活性的细胞代谢状态的调节剂。在这个项目中，我们想集中在精确的功能作用的AcuB，是由acuABC操纵子编码的B。枯草杆菌。这些数据将揭示，如果经典的脱乙酰酶的感官蛋白结合的调节是一个更普遍的监管系统。

Bacterial cells have to cope with rapidly changing environmental conditions, including alterations in nutrient availability. A way to sense the cellular metabolic state is the regulation of protein function by post-translational lysine acetylation. In bacteria, lysine acetylation is enzymatically catalyzed by lysine acetyltransferases (KATs) that use acetyl-CoA as donor molecule for the acetylation of lysine side chains. Acetylated lysine side chains can be deacetylated by deacetylases, which are classified into two groups, i.e. the classical Zn2+-dependent deacetylases (KDACs) and the NAD+-dependent sirtuins. Next to its role as donor molecule for acetylation, acetyl-CoA is a central metabolite. Acetyl-CoA can be, amongst other, generated by the activity of AMP-forming acetyl-CoA synthetases (Acs). This reaction is split into two half reactions. In the 1st half reaction, the adenylation reaction, acetate is activated by adenylation to yield acetyl-AMP. Subsequently, in the 2nd half reaction, acetyl-AMP is converted to the thioester acetyl-CoA (thioester formation). In the Gram-positive bacterial species Bacillus subtilis, the acetyl-CoA synthetase AcsA is encoded upstream from the acuABC-operon and is reversely transcribed. The acuABC-operon was originally identified to be involved in butanediol and acetoin catabolism. The function of the gene products of the acuABC operon are mechanistically only partially understood. The gene acuA encodes for a GNAT-type KAT to acetylate AcsA at a C-terminal lysine side chain, i.e. K549, and thereby inactivate AcsA activity. AcuC constitutes a classical Zn2+-dependent deacetylase. It is known that AcuC is capable to deacetylate and thereby (re-)activate AcsA. The exact molecular mechanisms underlying the inactivation of AcsA activity by AcuA mediated K549-acetylation are only marginally understood. The role of AcuB in the interplay of AcuA, AcuC and AcsA is totally unclear. Interestingly, other bacterial species such as Staphylococcus aureus share a similar gene organization. However, in S. aureus the operon encodes only for AcuA and AcuC but the acuB gene is missing. This suggests that AcuB plays a role that might be specific for some bacterial strains. We postulate that AcuB, which is completely functionally uncharacterized, might be an important regulator for this interplay, i.e. sensing the metabolic state, adjusting AcuA and/or AcuC activity to modulate AcsA activity. In B. subtilis, AcuB by binding to AcuC and/or AcuA and to molecules such as NAD+, AMP, ATP or CoA/acetyl-CoA might be the regulator that adapts the activity of the deactylase AcuC and/or the acetyltransferase AcuA to the cellular metabolic state enabling to coordinate AcsA activity. In this project we want to focus on the exact functional role of AcuB that is encoded by the acuABC-operon in B. subtilis. These data will reveal if regulation of classical deacetylases by binding to sensory proteins is a more general regulatory system.