Cargando…

Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F(420)-0 in Mycobacteria

F(420) is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study...

Descripción completa

Detalles Bibliográficos
Autores principales: Grinter, Rhys, Ney, Blair, Brammananth, Rajini, Barlow, Christopher K., Cordero, Paul R. F., Gillett, David L., Izoré, Thierry, Cryle, Max J., Harold, Liam K., Cook, Gregory M., Taiaroa, George, Williamson, Deborah A., Warden, Andrew C., Oakeshott, John G., Taylor, Matthew C., Crellin, Paul K., Jackson, Colin J., Schittenhelm, Ralf B., Coppel, Ross L., Greening, Chris
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253369/
https://www.ncbi.nlm.nih.gov/pubmed/32430409
http://dx.doi.org/10.1128/mSystems.00389-20
_version_ 1783539322366984192
author Grinter, Rhys
Ney, Blair
Brammananth, Rajini
Barlow, Christopher K.
Cordero, Paul R. F.
Gillett, David L.
Izoré, Thierry
Cryle, Max J.
Harold, Liam K.
Cook, Gregory M.
Taiaroa, George
Williamson, Deborah A.
Warden, Andrew C.
Oakeshott, John G.
Taylor, Matthew C.
Crellin, Paul K.
Jackson, Colin J.
Schittenhelm, Ralf B.
Coppel, Ross L.
Greening, Chris
author_facet Grinter, Rhys
Ney, Blair
Brammananth, Rajini
Barlow, Christopher K.
Cordero, Paul R. F.
Gillett, David L.
Izoré, Thierry
Cryle, Max J.
Harold, Liam K.
Cook, Gregory M.
Taiaroa, George
Williamson, Deborah A.
Warden, Andrew C.
Oakeshott, John G.
Taylor, Matthew C.
Crellin, Paul K.
Jackson, Colin J.
Schittenhelm, Ralf B.
Coppel, Ross L.
Greening, Chris
author_sort Grinter, Rhys
collection PubMed
description F(420) is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F(420) in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F(420) biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F(420) biosynthesis. Analysis of F(420) biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F(420)-0. To determine the structural basis of dehydro-F(420)-0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo-, substrate-, and product-bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic precomplex of this enzyme and demonstrate that dehydro-F(420)-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F(420) in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance. IMPORTANCE Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F(420), and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F(420) is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F(420) in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F(420), determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F(420).
format Online
Article
Text
id pubmed-7253369
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher American Society for Microbiology
record_format MEDLINE/PubMed
spelling pubmed-72533692020-06-08 Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F(420)-0 in Mycobacteria Grinter, Rhys Ney, Blair Brammananth, Rajini Barlow, Christopher K. Cordero, Paul R. F. Gillett, David L. Izoré, Thierry Cryle, Max J. Harold, Liam K. Cook, Gregory M. Taiaroa, George Williamson, Deborah A. Warden, Andrew C. Oakeshott, John G. Taylor, Matthew C. Crellin, Paul K. Jackson, Colin J. Schittenhelm, Ralf B. Coppel, Ross L. Greening, Chris mSystems Research Article F(420) is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F(420) in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F(420) biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F(420) biosynthesis. Analysis of F(420) biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F(420)-0. To determine the structural basis of dehydro-F(420)-0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo-, substrate-, and product-bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic precomplex of this enzyme and demonstrate that dehydro-F(420)-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F(420) in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance. IMPORTANCE Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F(420), and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F(420) is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F(420) in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F(420), determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F(420). American Society for Microbiology 2020-05-19 /pmc/articles/PMC7253369/ /pubmed/32430409 http://dx.doi.org/10.1128/mSystems.00389-20 Text en Copyright © 2020 Grinter et al. https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Research Article
Grinter, Rhys
Ney, Blair
Brammananth, Rajini
Barlow, Christopher K.
Cordero, Paul R. F.
Gillett, David L.
Izoré, Thierry
Cryle, Max J.
Harold, Liam K.
Cook, Gregory M.
Taiaroa, George
Williamson, Deborah A.
Warden, Andrew C.
Oakeshott, John G.
Taylor, Matthew C.
Crellin, Paul K.
Jackson, Colin J.
Schittenhelm, Ralf B.
Coppel, Ross L.
Greening, Chris
Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F(420)-0 in Mycobacteria
title Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F(420)-0 in Mycobacteria
title_full Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F(420)-0 in Mycobacteria
title_fullStr Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F(420)-0 in Mycobacteria
title_full_unstemmed Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F(420)-0 in Mycobacteria
title_short Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F(420)-0 in Mycobacteria
title_sort cellular and structural basis of synthesis of the unique intermediate dehydro-f(420)-0 in mycobacteria
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253369/
https://www.ncbi.nlm.nih.gov/pubmed/32430409
http://dx.doi.org/10.1128/mSystems.00389-20
work_keys_str_mv AT grinterrhys cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT neyblair cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT brammananthrajini cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT barlowchristopherk cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT corderopaulrf cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT gillettdavidl cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT izorethierry cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT crylemaxj cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT haroldliamk cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT cookgregorym cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT taiaroageorge cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT williamsondeboraha cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT wardenandrewc cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT oakeshottjohng cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT taylormatthewc cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT crellinpaulk cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT jacksoncolinj cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT schittenhelmralfb cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT coppelrossl cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria
AT greeningchris cellularandstructuralbasisofsynthesisoftheuniqueintermediatedehydrof4200inmycobacteria