Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F ₄₂₀ -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 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.

Diversification by CofC and Control by CofD Govern Biosynthesis and Evolution of Coenzyme F 420 and Its Derivative 3PG-F 420

“…A follow-up study delivered further evidence supporting the biosynthetic route via DF 420 in mycobacteria. The authors showed the formation of DF 420 from PEP in cell extracts of M. smegmatis and could reveal strong binding of DF 420 to the active site of FbiA (CofD) by X-ray crystallographic studies of the enzyme ( 34 ).…”

Modulation of riboflavin biosynthesis and utilization in mycobacteria

“…Step 7. Through distinct enzymatic reactions mediated by FbiC, FbiB and FbiA, 5-A-RU can also be converted to F 420 (57). DMRL and its derivatives can act as MAIT cell activators.…”

Role of Premycofactocin Synthase in Growth, Microaerophilic Adaptation, and Metabolism of Mycobacterium tuberculosis

“…S1A in the supplemental material). Based on prior experimental evidence ( 14 – 17 ), it is expected that the constructed M. tuberculosis Δ mftD is most likely mycofactocin deficient, thereby resembling impaired F 420 synthesis in a Mycobacterium smegmatis strain lacking either of the F 420 biosynthesis genes ( 19 ). As such, disruption of mftD had no impact on M. tuberculosis H37Rv growth on glycerol-supplemented Middlebrook 7H9 (m7H9) medium under well-aerated conditions ( Fig.…”

“…The mftD -encoded product catalyzes the crucial step in formation of premycofactocin—a penultimate biosynthetic intermediate, which could oxidize NADH, and its calculated midpoint potential is ≈−255 mV ( 14 ). Another noncanonical cofactor is F 420 , which is generated through the aerobic route with relatively lower redox potential in mycobacterial species but facilitates M. tuberculosis microaerophilic adaptation ( 19 , 40 – 42 ). F 420 cofactor recycling in methanogenic (anaerobic) archaea is achieved by F 420 reductase (Fno), which transfers electrons between F 420 and NADPH pools.…”