title: Active acetyl-CoA synthase from Clostridium thermoaceticum obtained by cloning and heterologous expression of acsAB in Escherichia coli.
abstract: 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.
authors.affiliation: Departments of Chemistry and Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.
authors.forename: H K
authors.initials: HK
authors.lastname: Loke
authors.forename: G N
authors.initials: GN
authors.lastname: Bennett
authors.forename: P A
authors.initials: PA
authors.lastname: Lindahl
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002245:Carbon Dioxide / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009532:Nickel / Q000032:analysis; D010640:Phenothiazines / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D009097:Multienzyme Complexes; D010640:Phenothiazines; D011993:Recombinant Fusion Proteins; D002245:Carbon Dioxide; D000105:Acetyl Coenzyme A; D009532:Nickel; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase; C009469:thionine