Active acetyl-CoA synthase from Clostridium thermoaceticum obtained by cloning and heterologous expression of acsAB in Escherichia coli
Active acetyl-CoA synthase from Clostridium thermoaceticum obtained by cloning and heterologous expression of acsAB in Escherichia coli
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DOI：
10.1073/pnas.220404397
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发表时间：
2000-11-07
影响因子：
11.1
通讯作者：
Lindahl, PA
中科院分区：
文献类型：
--
作者：
Loke, HK;Bennett, GN;Lindahl, PA
来自热醋酸梭菌的乙酰辅酶A合成酶(ACS(Ct))是一个α(2)β(2)四聚体，含有两个新的Ni-X-Fe4S4活性中心(A和C簇)和一个标准的Fe4S4簇(B簇)。将编码该酶的acsA和acsB基因克隆到大肠杆菌JM109中，并在添加镍的厌氧条件下37℃超量表达。分离到的重组His标记蛋白(AcsAB)具有与ACS(Ct)基本相同的特征。其中Ni已从A簇中移除。AcsAB在非变性凝胶中呈单一条带迁移，亚基摩尔比为1：1，亚基组成为1.0-1.6Ni/α和14-22Fe/α。AcsAB具有100-250个单位/mg的CO氧化活性，但没有CO/乙酰-CoA交换活性。硫代氧化和CO还原的AcsAB的电子吸收光谱与ACs(Ct)相似，具有典型的氧化还原活性Fe4S4团簇的特征。部分氧化和CO还原的AcsAB显示出g值和低自旋强度的EPR信号，这些信号与ACS(Ct)的B团簇的培育态和C团簇的C-Red1和C-red2态的EPR信号难以区分。在NiCl2中过夜后，产生的重组酶(ACS(EC))具有0.06-0.25个单位/毫克的交换活性。这些值中的最高值是典型的完全活动的急性冠脉综合征(CT)。当用CO还原时，ACs(EC)的EPR信号与富镍的ACs(Ct)的NiFeC信号难以区分。观察到不同制剂之间活性和信号强度的差异。讨论了在大肠杆菌中组装这些金属中心的问题。
Acetyl-CoA synthase from Clostridium thermoaceticum (ACS(Ct)) is an alpha (2)beta (2) tetramer containing two novel Ni-X-Fe4S4 active sites (the A and C clusters) and a standard Fe4S4 cluster (the B cluster). The acsA and acsB genes encoding the enzyme were cloned into Escherichia coli strain JM109 and overexpressed at 37 degreesC under anaerobic conditions with Ni supplementation. The isolated recombinant His-tagged protein (AcsAB) exhibited characteristics essentially indistinguishable from those of ACS(Ct). from which Ni had been removed from the A cluster. AcsAB migrated through nondenaturing electrophoretic gels as a single band and contained a 1:1 molar ratio of subunits and 1.0-1.6 Ni/alpha beta and 14-22 Fe/alpha beta. AcsAB exhibited 100-250 units/mg CO oxidation activity but no CO/acetyl-CoA exchange activity. Electronic absorption spectra of thionin-oxidized and CO-reduced AcsAB were similar to those of ACS(Ct), with features typical of redox-active Fe4S4 clusters. Partially oxidized and CO-reduced AcsAB exhibited EPR signals with g values and low spin intensities indistinguishable from those of the Bred State of the B cluster and the C-red1 and C-red2 states of the C cluster of ACS(Ct). Upon overnight exposure to NiCl2, the resulting recombinant enzyme (ACS(Ec)) developed 0.06-0.25 units/mg exchange activity. The highest of these values is typical of fully active ACS(Ct). When reduced with CO, ACS(Ec) exhibited an EPR signal indistinguishable from the NiFeC signal of Ni-replete ACS(Ct). Variability of activities and signal intensities were observed among different preparations. Issues involving the assembly of these metal centers in E. coli are discussed.