Active acetyl-CoA synthase from Clostridium thermoaceticum obtained by cloning and heterologous expression of acsAB in Escherichia coli

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 acs B genes encoding the enzyme were cloned into Escherichia coli strain JM109 and overexpressed at 37 degreesC under anaerobic conditions with Ni supplem entation. The isolated recombinant His-tagged protein (AcsAB) exhibited cha racteristics essentially indistinguishable from those of ACS(Ct). from whic h Ni had been removed from the A cluster. AcsAB migrated through nondenatur ing electrophoretic gels as a single band and contained a 1:1 molar ratio o f subunits and 1.0-1.6 Ni/alpha beta and 14-22 Fe/alpha beta. AcsAB exhibit ed 100-250 units/mg CO oxidation activity but no CO/acetyl-CoA exchange act ivity. Electronic absorption spectra of thionin-oxidized and CO-reduced Acs AB were similar to those of ACS(Ct), with features typical of redox-active Fe4S4 clusters. Partially oxidized and CO-reduced AcsAB exhibited EPR signa ls with g values and low spin intensities indistinguishable from those of t he Bred State of the B cluster and the C-red1 and C-red2 states of the C cl uster of ACS(Ct). Upon overnight exposure to NiCl2, the resulting recombina nt enzyme (ACS(Ec)) developed 0.06-0.25 units/mg exchange activity. The hig hest 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 we re observed among different preparations. Issues involving the assembly of these metal centers in E. coli are discussed.