A defining feature of mycobacterial redox metabolism is the use of an unusual deazaflavin cofactor, F 420 . 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 F 420 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 F 420 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 F 420 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 F 420 H 2 -dependent reductases directly reduce these compounds by a mechanism consistent with hydride transfer. Moreover, M. smegmatis strains unable to make F 420 H 2 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 F 420 loss of function (e.g., redox imbalance) rather than loss of a detoxification system. Together, these data show that F 420 enhances antimicrobial resistance in mycobacteria and suggest that one function of the F 420 H 2 -dependent reductases is to broaden the range of natural products that mycobacteria and possibly other environmental actinobacteria can reductively detoxify. IMPORTANCEThis study reveals that a unique microbial cofactor, F 420 , is critical for antimicrobial resistance in the environmental actinobacterium Mycobacterium smegmatis. We show that a superfamily of redox enzymes, the F 420 H 2 -dependent reductases, can reduce diverse antimicrobials in vitro and in vivo. M. smegmatis strains unable to make or reduce F 420 become sensitive to inhibition by these antimicrobial compounds. This suggests that mycobacteria have harnessed the unique properties of F 420 to reduce structurally diverse antimicrobials as part of the antibiotic arms race. The F 420 H 2 -dependent reductases that facilitate this process represent a new class of antimicrobial-detoxifying enzymes with potential applications in bioremediation and biocatalysis.

An unusual aspect of actinobacterial metabolism is the use of the redox cofactor F420. Studies have shown that actinobacterial F420H2-dependent reductases promiscuously hydrogenate diverse organic compounds in biodegradative and biosynthetic processes. These enzymes therefore represent promising candidates for next-generation industrial biocatalysts. In this work, we undertook the first broad survey of these enzymes as potential industrial biocatalysts by exploring the extent, as well as mechanistic and structural bases, of their substrate promiscuity. We expressed and purified 11 enzymes from seven subgroups of the flavin/deazaflavin oxidoreductase (FDOR) superfamily (A1, A2, A3, B1, B2, B3, B4) from the model soil actinobacterium Mycobacterium smegmatis. These enzymes reduced compounds from six chemical classes, including fundamental monocycles such as a cyclohexenone, a dihydropyran, and pyrones, as well as more complex quinone, coumarin, and arylmethane compounds. Substrate range and reduction rates varied between the enzymes, with the A1, A3, and B1 groups exhibiting greatest promiscuity. Molecular docking studies suggested that structurally diverse compounds are accommodated in the large substrate-binding pocket of the most promiscuous FDOR through hydrophobic interactions with conserved aromatic residues and the isoalloxazine headgroup of F420H2. Liquid chromatography-mass spectrometry (LC/MS) and gas chromatography-mass spectrometry (GC/MS) analysis of derivatized reaction products showed reduction occurred through a common mechanism involving hydride transfer from F420H- to the electron-deficient alkene groups of substrates. Reduction occurs when the hydride donor (C5 of F420H-) is proximal to the acceptor (electrophilic alkene of the substrate). These findings suggest that engineered actinobacterial F420H2-dependent reductases are promising novel biocatalysts for the facile transformation of a wide range of α,β-unsaturated compounds.