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A Novel NADP-Dependent Formate Dehydrogenase From the Hyperthermophilic Archaeon Thermococcus onnurineus NA1

The genome of the hyperthermophilic archaeon Thermococcus onnurineus NA1 contains three copies of the formate dehydrogenase (FDH) gene, fdh1, fdh2, and fdh3. Previously, we reported that fdh2, clustered with genes encoding the multimeric membrane-bound hydrogenase and cation/proton antiporter, was e...

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Autores principales: Yang, Ji-in, Lee, Seong Hyuk, Ryu, Ji-Young, Lee, Hyun Sook, Kang, Sung Gyun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8965080/
https://www.ncbi.nlm.nih.gov/pubmed/35369452
http://dx.doi.org/10.3389/fmicb.2022.844735
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author Yang, Ji-in
Lee, Seong Hyuk
Ryu, Ji-Young
Lee, Hyun Sook
Kang, Sung Gyun
author_facet Yang, Ji-in
Lee, Seong Hyuk
Ryu, Ji-Young
Lee, Hyun Sook
Kang, Sung Gyun
author_sort Yang, Ji-in
collection PubMed
description The genome of the hyperthermophilic archaeon Thermococcus onnurineus NA1 contains three copies of the formate dehydrogenase (FDH) gene, fdh1, fdh2, and fdh3. Previously, we reported that fdh2, clustered with genes encoding the multimeric membrane-bound hydrogenase and cation/proton antiporter, was essential for formate-dependent growth with H(2) production. However, the functionality of the other two FDH-coding genes has not yet been elucidated. Herein, we purified and characterized cytoplasmic Fdh3 to understand its functionality. The purified Fdh3 was identified to be composed of a tungsten-containing catalytic subunit (Fdh3A), an NAD(P)-binding protein (Fdh3B), and two Fe-S proteins (Fdh3G1 and Fdh3G2). Fdh3 oxidized formate with specific activities of 241.7 U/mg and 77.4 U/mg using methyl viologen and NADP(+) as electron acceptors, respectively. While most FDHs exhibited NAD(+)-dependent formate oxidation activity, the Fdh3 of T. onnurineus NA1 showed a strong preference for NADP(+) over NAD(+) as a cofactor. The catalytic efficiency (k(cat)/K(m)) of Fdh3 for NADP(+) was measured to be 5,281 mM(−1) s(−1), which is the highest among NADP-dependent FDHs known to date. Structural modeling suggested that Arg(204) and Arg(205) of Fdh3B may contribute to the stabilization of the 2′-phosphate of NADP(H). Fdh3 could also use ferredoxin as an electron acceptor to oxidize formate with a specific activity of 0.83 U/mg. Furthermore, Fdh3 showed CO(2) reduction activity using reduced ferredoxin or NADPH as an electron donor with a specific activity of 0.73 U/mg and 1.0 U/mg, respectively. These results suggest a functional role of Fdh3 in disposing of reducing equivalents by mediating electron transfer between formate and NAD(P)H or ferredoxin.
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spelling pubmed-89650802022-03-31 A Novel NADP-Dependent Formate Dehydrogenase From the Hyperthermophilic Archaeon Thermococcus onnurineus NA1 Yang, Ji-in Lee, Seong Hyuk Ryu, Ji-Young Lee, Hyun Sook Kang, Sung Gyun Front Microbiol Microbiology The genome of the hyperthermophilic archaeon Thermococcus onnurineus NA1 contains three copies of the formate dehydrogenase (FDH) gene, fdh1, fdh2, and fdh3. Previously, we reported that fdh2, clustered with genes encoding the multimeric membrane-bound hydrogenase and cation/proton antiporter, was essential for formate-dependent growth with H(2) production. However, the functionality of the other two FDH-coding genes has not yet been elucidated. Herein, we purified and characterized cytoplasmic Fdh3 to understand its functionality. The purified Fdh3 was identified to be composed of a tungsten-containing catalytic subunit (Fdh3A), an NAD(P)-binding protein (Fdh3B), and two Fe-S proteins (Fdh3G1 and Fdh3G2). Fdh3 oxidized formate with specific activities of 241.7 U/mg and 77.4 U/mg using methyl viologen and NADP(+) as electron acceptors, respectively. While most FDHs exhibited NAD(+)-dependent formate oxidation activity, the Fdh3 of T. onnurineus NA1 showed a strong preference for NADP(+) over NAD(+) as a cofactor. The catalytic efficiency (k(cat)/K(m)) of Fdh3 for NADP(+) was measured to be 5,281 mM(−1) s(−1), which is the highest among NADP-dependent FDHs known to date. Structural modeling suggested that Arg(204) and Arg(205) of Fdh3B may contribute to the stabilization of the 2′-phosphate of NADP(H). Fdh3 could also use ferredoxin as an electron acceptor to oxidize formate with a specific activity of 0.83 U/mg. Furthermore, Fdh3 showed CO(2) reduction activity using reduced ferredoxin or NADPH as an electron donor with a specific activity of 0.73 U/mg and 1.0 U/mg, respectively. These results suggest a functional role of Fdh3 in disposing of reducing equivalents by mediating electron transfer between formate and NAD(P)H or ferredoxin. Frontiers Media S.A. 2022-03-15 /pmc/articles/PMC8965080/ /pubmed/35369452 http://dx.doi.org/10.3389/fmicb.2022.844735 Text en Copyright © 2022 Yang, Lee, Ryu, Lee and Kang. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Yang, Ji-in
Lee, Seong Hyuk
Ryu, Ji-Young
Lee, Hyun Sook
Kang, Sung Gyun
A Novel NADP-Dependent Formate Dehydrogenase From the Hyperthermophilic Archaeon Thermococcus onnurineus NA1
title A Novel NADP-Dependent Formate Dehydrogenase From the Hyperthermophilic Archaeon Thermococcus onnurineus NA1
title_full A Novel NADP-Dependent Formate Dehydrogenase From the Hyperthermophilic Archaeon Thermococcus onnurineus NA1
title_fullStr A Novel NADP-Dependent Formate Dehydrogenase From the Hyperthermophilic Archaeon Thermococcus onnurineus NA1
title_full_unstemmed A Novel NADP-Dependent Formate Dehydrogenase From the Hyperthermophilic Archaeon Thermococcus onnurineus NA1
title_short A Novel NADP-Dependent Formate Dehydrogenase From the Hyperthermophilic Archaeon Thermococcus onnurineus NA1
title_sort novel nadp-dependent formate dehydrogenase from the hyperthermophilic archaeon thermococcus onnurineus na1
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8965080/
https://www.ncbi.nlm.nih.gov/pubmed/35369452
http://dx.doi.org/10.3389/fmicb.2022.844735
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