Active acetyl-CoA synthase from Clostridium thermoaceticum obtained by cloning and heterologous expression of acsAB in Escherichia coli
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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.
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CODH/ACS was functional in both CO oxidation and CO/acetyl-CoA exchange activities after heterologous expression in and purification from E. coli.
“In a later study, it could be shown that the CODH/ACS was functional in both CO oxidation and CO/acetyl-CoA exchange activities after heterologous expression in and purification from E. coli (Loke et al., 2000).”
From Gas Fermentation—A Flexible Platform for Commercial Scale Production of Low-Carbon-Fuels and Chemicals from Waste and Renewable Feedstocks (2016) · cites this paper as Loke et al., 2000
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Liew, F., Martin, M. E., Tappel, R. C., Heijstra, B. D., Mihalcea, C., & Köpke, M. (2016) · Frontiers in Microbiology · doi:10.3389/fmicb.2016.00694 · Cited by 485 · see citing works
- Authors
- Loke, H.-K., Bennett, G. N., & Lindahl, P. A.
- Affiliations
- Rice University (US)
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2000-10-24
- Volume
- 97
- Issue
- 23
- Pages
- 12530-12535
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC18798
- OpenAlex
- W1980817415
- Cited by
- 33
- References
- 63
- Citations by year
- 2026: 1 · 2024: 1 · 2022: 2 · 2021: 1 · 2020: 1 · 2018: 3 · 2017: 2 · 2016: 2 · 2015: 2 · 2014: 1 · 2013: 1 · 2012: 1
- FWCI
- 1.33
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- OakTrust (Texas A&M University Libraries) (submittedVersion)
- Funders
- National Institute of General Medical Sciences (R01GM046441, R56GM046441, GM 46441)
- Topics
- Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Metalloenzymes and iron-sulfur proteins
- Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
- Physical Sciences › Chemistry › Inorganic Chemistry › Metal-Catalyzed Oxygenation Mechanisms
- Keywords
- acetyl-CoA synthase, Clostridium thermoaceticum, heterologous expression in Escherichia coli, Fe4S4 cluster, A-cluster, C-cluster, electron spin resonance, recombinant enzyme
- MeSH terms
- Acetyl Coenzyme A, Aldehyde Oxidoreductases, Carbon Dioxide, Catalysis, Cloning, Molecular, Clostridium, Enzyme Activation, Escherichia coli, Multienzyme Complexes, Nickel, Phenothiazines, Recombinant Fusion Proteins, Gene Expression
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