Cargando…
Minimal Pathway for the Regeneration of Redox Cofactors
[Image: see text] Effective metabolic pathways are essential for the construction of in vitro systems mimicking the biochemical complexity of living cells. Such pathways require the inclusion of a metabolic branch that ensures the availability of reducing equivalents. Here, we built a minimal enzyma...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2021
|
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8717395/ https://www.ncbi.nlm.nih.gov/pubmed/34984417 http://dx.doi.org/10.1021/jacsau.1c00406 |
_version_ | 1784624523216158720 |
---|---|
author | Partipilo, Michele Ewins, Eleanor J. Frallicciardi, Jacopo Robinson, Tom Poolman, Bert Slotboom, Dirk Jan |
author_facet | Partipilo, Michele Ewins, Eleanor J. Frallicciardi, Jacopo Robinson, Tom Poolman, Bert Slotboom, Dirk Jan |
author_sort | Partipilo, Michele |
collection | PubMed |
description | [Image: see text] Effective metabolic pathways are essential for the construction of in vitro systems mimicking the biochemical complexity of living cells. Such pathways require the inclusion of a metabolic branch that ensures the availability of reducing equivalents. Here, we built a minimal enzymatic pathway confinable in the lumen of liposomes, in which the redox status of the nicotinamide cofactors NADH and NADPH is controlled by an externally provided formate. Formic acid permeates the membrane where a luminal formate dehydrogenase uses NAD(+) to form NADH and carbon dioxide. Carbon dioxide diffuses out of the liposomes, leaving only the reducing equivalents in the lumen. A soluble transhydrogenase subsequently utilizes NADH for reduction of NADP(+) thereby making NAD(+) available again for the first reaction. The pathway is functional in liposomes ranging from a few hundred nanometers in diameter (large unilamellar vesicles) up to several tens of micrometers (giant unilamellar vesicles) and remains active over a period of 7 days. We demonstrate that the downstream biochemical process of reduction of glutathione disulfide can be driven by the transfer of reducing equivalents from formate via NAD(P)H, thereby providing a versatile set of electron donors for reductive metabolism. |
format | Online Article Text |
id | pubmed-8717395 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-87173952022-01-03 Minimal Pathway for the Regeneration of Redox Cofactors Partipilo, Michele Ewins, Eleanor J. Frallicciardi, Jacopo Robinson, Tom Poolman, Bert Slotboom, Dirk Jan JACS Au [Image: see text] Effective metabolic pathways are essential for the construction of in vitro systems mimicking the biochemical complexity of living cells. Such pathways require the inclusion of a metabolic branch that ensures the availability of reducing equivalents. Here, we built a minimal enzymatic pathway confinable in the lumen of liposomes, in which the redox status of the nicotinamide cofactors NADH and NADPH is controlled by an externally provided formate. Formic acid permeates the membrane where a luminal formate dehydrogenase uses NAD(+) to form NADH and carbon dioxide. Carbon dioxide diffuses out of the liposomes, leaving only the reducing equivalents in the lumen. A soluble transhydrogenase subsequently utilizes NADH for reduction of NADP(+) thereby making NAD(+) available again for the first reaction. The pathway is functional in liposomes ranging from a few hundred nanometers in diameter (large unilamellar vesicles) up to several tens of micrometers (giant unilamellar vesicles) and remains active over a period of 7 days. We demonstrate that the downstream biochemical process of reduction of glutathione disulfide can be driven by the transfer of reducing equivalents from formate via NAD(P)H, thereby providing a versatile set of electron donors for reductive metabolism. American Chemical Society 2021-11-12 /pmc/articles/PMC8717395/ /pubmed/34984417 http://dx.doi.org/10.1021/jacsau.1c00406 Text en © 2021 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Partipilo, Michele Ewins, Eleanor J. Frallicciardi, Jacopo Robinson, Tom Poolman, Bert Slotboom, Dirk Jan Minimal Pathway for the Regeneration of Redox Cofactors |
title | Minimal Pathway for the Regeneration of Redox Cofactors |
title_full | Minimal Pathway for the Regeneration of Redox Cofactors |
title_fullStr | Minimal Pathway for the Regeneration of Redox Cofactors |
title_full_unstemmed | Minimal Pathway for the Regeneration of Redox Cofactors |
title_short | Minimal Pathway for the Regeneration of Redox Cofactors |
title_sort | minimal pathway for the regeneration of redox cofactors |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8717395/ https://www.ncbi.nlm.nih.gov/pubmed/34984417 http://dx.doi.org/10.1021/jacsau.1c00406 |
work_keys_str_mv | AT partipilomichele minimalpathwayfortheregenerationofredoxcofactors AT ewinseleanorj minimalpathwayfortheregenerationofredoxcofactors AT frallicciardijacopo minimalpathwayfortheregenerationofredoxcofactors AT robinsontom minimalpathwayfortheregenerationofredoxcofactors AT poolmanbert minimalpathwayfortheregenerationofredoxcofactors AT slotboomdirkjan minimalpathwayfortheregenerationofredoxcofactors |