Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F<sub>420</sub>-0 in Mycobacteria

ABSTRACT F420 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...

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Autores principales:	Rhys Grinter, Blair Ney, Rajini Brammananth, Christopher K. Barlow, Paul R. F. Cordero, David L. Gillett, Thierry Izoré, Max J. Cryle, Liam K. Harold, Gregory M. Cook, George Taiaroa, Deborah A. Williamson, Andrew C. Warden, John G. Oakeshott, Matthew C. Taylor, Paul K. Crellin, Colin J. Jackson, Ralf B. Schittenhelm, Ross L. Coppel, Chris Greening
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	cofactor biosynthesis deazaflavin F420 Mycobacterium Mycobacterium smegmatis structural biology Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/63558d0234fb43f688726db29ee00c9a
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spelling	oai:doaj.org-article:63558d0234fb43f688726db29ee00c9a2021-12-02T19:47:39ZCellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F<sub>420</sub>-0 in Mycobacteria10.1128/mSystems.00389-202379-5077https://doaj.org/article/63558d0234fb43f688726db29ee00c9a2020-06-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00389-20https://doaj.org/toc/2379-5077ABSTRACT F420 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 F420 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 F420 biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F420 biosynthesis. Analysis of F420 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-F420-0. To determine the structural basis of dehydro-F420-0 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-F420-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F420 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, F420, and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F420 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 F420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F420, 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 F420.Rhys GrinterBlair NeyRajini BrammananthChristopher K. BarlowPaul R. F. CorderoDavid L. GillettThierry IzoréMax J. CryleLiam K. HaroldGregory M. CookGeorge TaiaroaDeborah A. WilliamsonAndrew C. WardenJohn G. OakeshottMatthew C. TaylorPaul K. CrellinColin J. JacksonRalf B. SchittenhelmRoss L. CoppelChris GreeningAmerican Society for Microbiologyarticlecofactor biosynthesisdeazaflavinF420MycobacteriumMycobacterium smegmatisstructural biologyMicrobiologyQR1-502ENmSystems, Vol 5, Iss 3 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	cofactor biosynthesis deazaflavin F420 Mycobacterium Mycobacterium smegmatis structural biology Microbiology QR1-502
spellingShingle	cofactor biosynthesis deazaflavin F420 Mycobacterium Mycobacterium smegmatis structural biology Microbiology QR1-502 Rhys Grinter Blair Ney Rajini Brammananth Christopher K. Barlow Paul R. F. Cordero David L. Gillett Thierry Izoré Max J. Cryle Liam K. Harold Gregory M. Cook George Taiaroa Deborah A. Williamson Andrew C. Warden John G. Oakeshott Matthew C. Taylor Paul K. Crellin Colin J. Jackson Ralf B. Schittenhelm Ross L. Coppel Chris Greening Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F<sub>420</sub>-0 in Mycobacteria
description	ABSTRACT F420 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 F420 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 F420 biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F420 biosynthesis. Analysis of F420 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-F420-0. To determine the structural basis of dehydro-F420-0 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-F420-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F420 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, F420, and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F420 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 F420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F420, 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 F420.
format	article
author	Rhys Grinter Blair Ney Rajini Brammananth Christopher K. Barlow Paul R. F. Cordero David L. Gillett Thierry Izoré Max J. Cryle Liam K. Harold Gregory M. Cook George Taiaroa Deborah A. Williamson Andrew C. Warden John G. Oakeshott Matthew C. Taylor Paul K. Crellin Colin J. Jackson Ralf B. Schittenhelm Ross L. Coppel Chris Greening
author_facet	Rhys Grinter Blair Ney Rajini Brammananth Christopher K. Barlow Paul R. F. Cordero David L. Gillett Thierry Izoré Max J. Cryle Liam K. Harold Gregory M. Cook George Taiaroa Deborah A. Williamson Andrew C. Warden John G. Oakeshott Matthew C. Taylor Paul K. Crellin Colin J. Jackson Ralf B. Schittenhelm Ross L. Coppel Chris Greening
author_sort	Rhys Grinter
title	Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F<sub>420</sub>-0 in Mycobacteria
title_short	Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F<sub>420</sub>-0 in Mycobacteria
title_full	Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F<sub>420</sub>-0 in Mycobacteria
title_fullStr	Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F<sub>420</sub>-0 in Mycobacteria
title_full_unstemmed	Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F<sub>420</sub>-0 in Mycobacteria
title_sort	cellular and structural basis of synthesis of the unique intermediate dehydro-f<sub>420</sub>-0 in mycobacteria
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/63558d0234fb43f688726db29ee00c9a
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Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F<sub>420</sub>-0 in Mycobacteria

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