Cited by 33Metalloenzymes and iron-sulfur proteinsEnzyme Structure and FunctionMetal-Catalyzed Oxygenation Mechanisms

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

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Loke, Huay-Keng, Bennett, George N., Lindahl, Paul A.·2000·Proceedings of the National Academy of Sciences

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.

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Crossref · 10.1073/pnas.220404397

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Authors
Loke, H.-K., Bennett, G. N., & Lindahl, P. A.
Affiliations
Rice University (US)
Venue
Proceedings of the National Academy of Sciences
Published
2000-10-24
Volume
97
Issue
23
Pages
12530-12535
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
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
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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