The Redox Cofactor F₄₂₀ Protects Mycobacteria from Diverse Antimicrobial Compounds and Mediates a Reductive...

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 after antimicrobial treatment, although 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 the 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. In 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.IMPORTANCE 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 vivoM. 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 antibiotic arms race. The F₄₂₀H₂-dependent reductases that facilitate this process represent a new class of antimicrobial-detoxifying enzymes with potential applications in bioremediation and biocatalysis.