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

AUTOR(ES)

Loke, Huay-Keng

FONTE

The National Academy of Sciences

RESUMO

Acetyl-CoA synthase from Clostridium thermoaceticum (ACSCt) is an α2β2 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 37oC under anaerobic conditions with Ni supplementation. The isolated recombinant His-tagged protein (AcsAB) exhibited characteristics essentially indistinguishable from those of ACSCt, 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/αβ and 14–22 Fe/αβ. 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 ACSCt, 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 Cred1 and Cred2 states of the C cluster of ACSCt. Upon overnight exposure to NiCl2, the resulting recombinant enzyme (ACSEc) developed 0.06–0.25 units/mg exchange activity. The highest of these values is typical of fully active ACSCt. When reduced with CO, ACSEc exhibited an EPR signal indistinguishable from the NiFeC signal of Ni-replete ACSCt. Variability of activities and signal intensities were observed among different preparations. Issues involving the assembly of these metal centers in E. coli are discussed.

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