CooC2/AcsF and Cfd1/Nbp35: maturation of complex Fe/S-clusters by MinD-type ATPases

CooC2/AcsF 和 Cfd1/Nbp35：MinD 型 ATP 酶对复杂 Fe/S 簇的成熟

• 批准号: 311061912
• 负责人: Professor Dr. Holger Dobbek
• 金额: --
• 依托单位: Arbeitsgruppe Strukturbiologie und Biochemie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/311061912?language=en
• 关键词: CooC2; AcsF; Cfd1; Nbp35; maturation

Anaerobic bacteria and archaea use homologous enzymes to convert CO2 and H2 to acetyl-CoA and methane, which determine since the dawn of life the global carbon cycle on earth. In recent years, we have discovered and investigated a Ni insertase for the central enzyme acetyl-CoA synthase (ACS). While one or more maturation enzymes are known for all other central Ni enzymes; so far no enzyme supporting the maturation of Ni,Ni-[4Fe4S] cluster, called Cluster A, has been described. We could show that the maturation protein CooC2, a MinD-family ATPase, forms a specific complex with the Ni-deficient ACS, that this protein complex binds two Ni ions with high affinity and finally that the active ACS is liberated by the addition of Mg-ATP to the complex. Bioinformatic analyzes suggest that the CooC ATPases can be divided into three subclasses: one subclass contains ATPases for the maturation of the [NiFe4S4] center of the carbon monoxide dehydrogenases; one subclass, with CooC2 as the first example described functionally, consists of ATPases responsible for the maturation of the ACS; the role of the third subclass is unknown. In the first funding period we want to investigate: (I) whether the homologous enzymes from archaea have the same function as the bacterial CooC2 protein. This is particularly interesting since the ACS from Archaea differs in size and association with CODH and CoFeSP from the bacterial enzyme: compared to the bacterial enzymes it misses the N-terminal domain, but is present in a multi-enzyme complex of 2.5 MDa. We want to investigate (II), how the high affinity for Ni is generated in the CooC2-apoACS complex and which role ATP plays in the maturation. In addition to apo- and holo-ACS, we can produce two stable maturation intermediates giving us the rare opportunity to follow the stepwise assembly of a complex Ni, Fe, S-cluster. Furthermore, (III) we want to compare the insertion strategy of CooC2 with a different type MinD metal-insertase, Cfd1/Nbp35, a key complex of cytosolic Fe/S cluster maturation machinery. In order to achieve the three objectives, integration in the SPP and the interaction with other groups of the SPP are indispensable.

厌氧细菌和古生菌使用同源酶将二氧化碳和氢气转化为乙酰辅酶A和甲烷，这决定了自生命诞生以来地球上的全球碳循环。近年来，我们发现并研究了中心酶乙酰辅酶A合成酶(ACS)的镍插入酶。虽然所有其他中心镍酶都有一个或多个成熟酶已知，但到目前为止还没有支持镍成熟的酶，但还没有描述过镍-[4Fe4S]簇，称为簇A。我们可以证明成熟蛋白CooC2是一个Mind家族的ATPase，它与缺镍的ACS形成一个特异的复合体，该蛋白复合体以高亲和力结合两个镍离子，最后通过向该复合体中添加镁-ATP来释放具有活性的ACS。生物信息学分析表明，CooC ATPase可以分为三个亚类：一个亚类包含一氧化碳脱氢酶[NiFe4S4]中心成熟的ATPase；一个亚类是第一个从功能上描述的CooC2亚类，由负责ACS成熟的ATPase组成；第三个亚类的作用未知。在第一个资助期，我们想要研究：(I)古生菌的同源酶是否具有与细菌CooC2蛋白相同的功能。这一点特别有趣，因为来自古生代的ACS在大小和与CODH和CoFeSP的结合方面不同于细菌酶：与细菌酶相比，它缺少N-末端结构域，但存在于2.5丙二醛的多酶复合体中。我们想研究(II)，如何在CooC2-apoACS复合体中产生对Ni的高亲和力，以及ATP在成熟过程中扮演的角色。除了apo-和holo-acs，我们还可以产生两个稳定的成熟中间体，这给了我们一个难得的机会来跟踪一个复杂的Ni，Fe，S-簇的逐步组装。此外，(Iii)我们希望将CooC2的插入策略与不同类型的金属插入酶Cfd1/Nbp35进行比较，Cfd1/Nbp35是胞内Fe/S簇成熟机制的关键复合体。为了实现这三个目标，最高人民大会党的一体化以及与最高人民大会党其他群体的互动必不可少。