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 language | English (US) |
|---|---|
| Article number | 148080 |
| Journal | Biochimica et Biophysica Acta - Bioenergetics |
| Volume | 1860 |
| Issue number | 11 |
| DOIs | |
| State | Published - Nov 1 2019 |
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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 journal › Article
}
TY - JOUR
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.
PY - 2019/11/1
Y1 - 2019/11/1
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
KW - Flavoprotein
KW - NADH oxidation
KW - Thermus thermophilus
KW - X-ray structure
UR - http://www.scopus.com/inward/record.url?scp=85073121218&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85073121218&partnerID=8YFLogxK
U2 - 10.1016/j.bbabio.2019.148080
DO - 10.1016/j.bbabio.2019.148080
M3 - Article
C2 - 31520616
AN - SCOPUS:85073121218
VL - 1860
JO - Biochimica et Biophysica Acta - Bioenergetics
JF - Biochimica et Biophysica Acta - Bioenergetics
SN - 0005-2728
IS - 11
M1 - 148080
ER -