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Conversion of NO2 to NO by reduced coenzyme F420 protects mycobacteria from nitrosative damage.

ABSTRACT: In mycobacteria, F(420), a deazaflavin derivative, acts as a hydride transfer coenzyme for an F(420)-specific glucose-6-phosphate dehydrogenase (Fgd). Physiologically relevant reactions in the mycobacteria that use Fgd-generated reduced F(420) (F(420)H(2)) are unknown. In this work, F(420)H(2) was found to be oxidized by NO only in the presence of oxygen. Further analysis demonstrated that NO(2), produced from NO and O(2), was the oxidant. UV-visible spectroscopic and NO-sensor-based analyses proved that F(420)H(2) reduced NO(2) to NO. This reaction could serve as a defense system for Mycobacterium tuberculosis, which is more sensitive to NO(2) than NO under aerobic conditions. Activated macrophages produce NO, which in acidified phagosomes is converted to NO(2). Hence, by converting NO(2) back to NO with F(420)H(2), M. tuberculosis could decrease the effectiveness of antibacterial action of macrophages; such defense would correspond to active tuberculosis conditions where the bacterium grows aerobically. This hypothesis was consistent with the observation that a mutant strain of Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis, which either did not produce or could not reduce F(420), was approximately 4-fold more sensitive to NO(2) than the wild-type strain. The phenomenon is reminiscent of the anticancer activity of gamma-tocopherol, which reduces NO(2) to NO and protects human cells from NO(2)-induced carcinogenesis.

SUBMITTER: Purwantini E

PROVIDER: S-EPMC2669391 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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Conversion of NO2 to NO by reduced coenzyme F420 protects mycobacteria from nitrosative damage.

Purwantini Endang E Mukhopadhyay Biswarup B

Proceedings of the National Academy of Sciences of the United States of America 20090326 15

In mycobacteria, F(420), a deazaflavin derivative, acts as a hydride transfer coenzyme for an F(420)-specific glucose-6-phosphate dehydrogenase (Fgd). Physiologically relevant reactions in the mycobacteria that use Fgd-generated reduced F(420) (F(420)H(2)) are unknown. In this work, F(420)H(2) was found to be oxidized by NO only in the presence of oxygen. Further analysis demonstrated that NO(2), produced from NO and O(2), was the oxidant. UV-visible spectroscopic and NO-sensor-based analyses pr ...[more]

PMID: 19325122

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Project description:A defining feature of mycobacterial redox metabolism is the use of an unusual deazaflavin cofactor, F420 This cofactor enhances the persistence of environmental and pathogenic mycobacteria, including following antimicrobial treatment, though the molecular basis for this remains to be understood. In this work, we explored our hypothesis that F420 enhances persistence by serving as a cofactor in antimicrobial-detoxifying enzymes. To test this, we performed a series of phenotypic, biochemical, and analytical chemistry studies in relation to the model soil bacterium Mycobacterium smegmatis Mutant strains unable to synthesize or reduce F420 were found to be more susceptible to a wide range of antibiotic and xenobiotic compounds. Compounds from three classes of antimicrobial compounds traditionally resisted by mycobacteria inhibited growth of F420 mutant strains at subnanomolar concentrations, namely furanocoumarins (e.g. methoxsalen), arylmethanes (e.g. malachite green), and quinone analogues (e.g. menadione). We demonstrated that promiscuous F420H2-dependent reductases directly reduce these compounds by a mechanism consistent with hydride transfer. Moreover, M. smegmatis strains unable to make F420H2 lost the capacity to reduce and detoxify representatives of the furanocoumarin and arylmethane compound classes in whole-cell assays. By contrast, mutant strains were only slightly more susceptible to clinical antimycobacterials and this appeared to be due to indirect effects of F420 loss-of-function (e.g. redox imbalance) rather than loss of a detoxification system. Together, these data show that F420 enhances antimicrobial resistance in mycobacteria and suggest that one function of the F420H2-dependent reductases is to broaden the range of natural products that mycobacteria and possibly other environmental actinobacteria can reductively detoxify.This study reveals that a unique microbial cofactor, F420, is critical for antimicrobial resistance in the environmental actinobacterium Mycobacterium smegmatis We show that a superfamily of redox enzymes, the F420H2-dependent reductases, can reduce diverse antimicrobials in vitro and in vivo M. smegmatis strains unable to make or reduce F420 become sensitive to inhibition by these antimicrobial compounds. This suggests that mycobacteria have harnessed the unique properties of F420 to reduce structurally diverse antimicrobials as part of the antibiotics arm race. The F420H2-dependent reductases that facilitate this process represent a new class of antimicrobial-detoxifying enzymes with potential applications in bioremediation and biocatalysis.

Project description: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.

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Conversion of NO2 to NO by reduced coenzyme F420 protects mycobacteria from nitrosative damage.

Publications

Conversion of NO2 to NO by reduced coenzyme F420 protects mycobacteria from nitrosative damage.

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