Active acetyl-CoA synthase from Clostridium thermoaceticum obtained by cloning and heterologous expression of acsAB in Escherichia coli.
- Additional Document Info
- View All
Acetyl-CoA synthase from Clostridium thermoaceticum (ACS(Ct)) is an alpha(2)beta(2) tetramer containing two novel Ni-X-Fe(4)S(4) active sites (the A and C clusters) and a standard Fe(4)S(4) cluster (the B cluster). The acsA and acsB genes encoding the enzyme were cloned into Escherichia coli strain JM109 and overexpressed at 37(o)C 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/alphabeta and 14-22 Fe/alphabeta. 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 Fe(4)S(4) clusters. Partially oxidized and CO-reduced AcsAB exhibited EPR signals with g values and low spin intensities indistinguishable from those of the B(red) state of the B cluster and the C(red1) and C(red2) states of the C cluster of ACS(Ct). Upon overnight exposure to NiCl(2), 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.
author list (cited authors)
Loke, H. K., Bennett, G. N., & Lindahl, P. A.
complete list of authors
Loke, HK||Bennett, GN||Lindahl, PA