Characterization and X-ray structure of the NADH-dependent coenzyme A disulfide reductase from Thermus thermophilus

Andrea M. Lencina, Juergen Koepke, Julia Preu, Cornelia Muenke, Robert B Gennis, Hartmut Michel, Lici A. Schurig-Briccio

Research output: Contribution to journalArticle

Abstract

The crystal structure of the enzyme previously characterized as a type-2 NADH:menaquinone oxidoreductase (NDH-2) from Thermus thermophilus has been solved at a resolution of 2.9 Å and revealed that this protein is, in fact, a coenzyme A-disulfide reductase (CoADR). Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Thermus thermophilus and is maintained in the reduced state by this enzyme (CoADR). Although the enzyme does exhibit NADH:menadione oxidoreductase activity expected for NDH-2 enzymes, the specific activity with CoAD as an electron acceptor is about 5-fold higher than with menadione. Furthermore, the crystal structure contains coenzyme A covalently linked Cys44, a catalytic intermediate (Cys44-S-S-CoA) reduced by NADH via the FAD cofactor. Soaking the crystals with menadione shows that menadione can bind to a site near the redox active FAD, consistent with the observed NADH:menadione oxidoreductase activity. CoADRs from other species were also examined and shown to have measurable NADH:menadione oxidoreductase activity. Although a common feature of this family of enzymes, no biological relevance is proposed. The CoADR from T. thermophilus is a soluble homodimeric enzyme. Expression of the recombinant TtCoADR at high levels in E. coli results in a small fraction that co-purifies with the membrane fraction, which was used previously to isolate the enzyme wrongly identified as a membrane-bound NDH-2. It is concluded that T. thermophilus does not contain an authentic NDH-2 component in its aerobic respiratory chain.

Original languageEnglish (US)
Article number148080
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume1860
Issue number11
DOIs
StatePublished - Nov 1 2019

Fingerprint

Thermus thermophilus
Vitamin K 3
NAD
Oxidoreductases
X-Rays
X rays
Coenzyme A
Enzymes
Flavin-Adenine Dinucleotide
Crystal structure
Enzymes and Coenzymes
Membranes
Vitamin K 2
Electron Transport
coenzyme A disulfide
Sulfhydryl Compounds
Escherichia coli
Oxidation-Reduction
Glutathione
Molecular Weight

Keywords

  • Coenzyme A disulfide reductase
  • Flavoprotein
  • NADH oxidation
  • Thermus thermophilus
  • X-ray structure

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

Cite this

Characterization and X-ray structure of the NADH-dependent coenzyme A disulfide reductase from Thermus thermophilus. / Lencina, Andrea M.; Koepke, Juergen; Preu, Julia; Muenke, Cornelia; Gennis, Robert B; Michel, Hartmut; Schurig-Briccio, Lici A.

In: Biochimica et Biophysica Acta - Bioenergetics, Vol. 1860, No. 11, 148080, 01.11.2019.

Research output: Contribution to journalArticle

Lencina, Andrea M. ; Koepke, Juergen ; Preu, Julia ; Muenke, Cornelia ; Gennis, Robert B ; Michel, Hartmut ; Schurig-Briccio, Lici A. / Characterization and X-ray structure of the NADH-dependent coenzyme A disulfide reductase from Thermus thermophilus. In: Biochimica et Biophysica Acta - Bioenergetics. 2019 ; Vol. 1860, No. 11.
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T1 - Characterization and X-ray structure of the NADH-dependent coenzyme A disulfide reductase from Thermus thermophilus

AU - Lencina, Andrea M.

AU - Koepke, Juergen

AU - Preu, Julia

AU - Muenke, Cornelia

AU - Gennis, Robert B

AU - Michel, Hartmut

AU - Schurig-Briccio, Lici A.

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N2 - The crystal structure of the enzyme previously characterized as a type-2 NADH:menaquinone oxidoreductase (NDH-2) from Thermus thermophilus has been solved at a resolution of 2.9 Å and revealed that this protein is, in fact, a coenzyme A-disulfide reductase (CoADR). Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Thermus thermophilus and is maintained in the reduced state by this enzyme (CoADR). Although the enzyme does exhibit NADH:menadione oxidoreductase activity expected for NDH-2 enzymes, the specific activity with CoAD as an electron acceptor is about 5-fold higher than with menadione. Furthermore, the crystal structure contains coenzyme A covalently linked Cys44, a catalytic intermediate (Cys44-S-S-CoA) reduced by NADH via the FAD cofactor. Soaking the crystals with menadione shows that menadione can bind to a site near the redox active FAD, consistent with the observed NADH:menadione oxidoreductase activity. CoADRs from other species were also examined and shown to have measurable NADH:menadione oxidoreductase activity. Although a common feature of this family of enzymes, no biological relevance is proposed. The CoADR from T. thermophilus is a soluble homodimeric enzyme. Expression of the recombinant TtCoADR at high levels in E. coli results in a small fraction that co-purifies with the membrane fraction, which was used previously to isolate the enzyme wrongly identified as a membrane-bound NDH-2. It is concluded that T. thermophilus does not contain an authentic NDH-2 component in its aerobic respiratory chain.

AB - The crystal structure of the enzyme previously characterized as a type-2 NADH:menaquinone oxidoreductase (NDH-2) from Thermus thermophilus has been solved at a resolution of 2.9 Å and revealed that this protein is, in fact, a coenzyme A-disulfide reductase (CoADR). Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Thermus thermophilus and is maintained in the reduced state by this enzyme (CoADR). Although the enzyme does exhibit NADH:menadione oxidoreductase activity expected for NDH-2 enzymes, the specific activity with CoAD as an electron acceptor is about 5-fold higher than with menadione. Furthermore, the crystal structure contains coenzyme A covalently linked Cys44, a catalytic intermediate (Cys44-S-S-CoA) reduced by NADH via the FAD cofactor. Soaking the crystals with menadione shows that menadione can bind to a site near the redox active FAD, consistent with the observed NADH:menadione oxidoreductase activity. CoADRs from other species were also examined and shown to have measurable NADH:menadione oxidoreductase activity. Although a common feature of this family of enzymes, no biological relevance is proposed. The CoADR from T. thermophilus is a soluble homodimeric enzyme. Expression of the recombinant TtCoADR at high levels in E. coli results in a small fraction that co-purifies with the membrane fraction, which was used previously to isolate the enzyme wrongly identified as a membrane-bound NDH-2. It is concluded that T. thermophilus does not contain an authentic NDH-2 component in its aerobic respiratory chain.

KW - Coenzyme A disulfide reductase

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KW - Thermus thermophilus

KW - X-ray structure

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