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

Content Provider	PubMed Central
Author	Loke, Huay-keng Bennett, George N. Lindahl, Paul A.
Copyright Year	2000
Abstract	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.
Starting Page	12530
File Format	PDF
ISSN	10916490
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	23
Volume Number	97
Language	English
Publisher	The National Academy of Sciences
Publisher Date	2000-11-07
Access Restriction	Open
Rights Holder	The National Academy of Sciences
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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