Cofactor F 420 plays critical roles in primary and secondary metabolism in a range of bacteria and archaea as a low-potential hydride transfer agent. It mediates a variety of important redox transformations involved in bacterial persistence, antibiotic biosynthesis, pro-drug activation and methanogenesis. However, the biosynthetic pathway for F 420 has not been fully elucidated: neither the enzyme that generates the putative intermediate 2-phospho- l -lactate, nor the function of the FMN-binding C-terminal domain of the γ-glutamyl ligase (FbiB) in bacteria are known. Here we present the structure of the guanylyltransferase FbiD and show that, along with its archaeal homolog CofC, it accepts phosphoenolpyruvate, rather than 2-phospho- l -lactate, as the substrate, leading to the formation of the previously uncharacterized intermediate dehydro-F 420 -0. The C-terminal domain of FbiB then utilizes FMNH 2 to reduce dehydro-F 420 -0, which produces mature F 420 species when combined with the γ-glutamyl ligase activity of the N-terminal domain. These new insights have allowed the heterologous production of F 420 from a recombinant F 420 biosynthetic pathway in Escherichia coli .

F 420 is a low-potential redox cofactor that mediates the transformations of a wide range of complex organic compounds. Considered one of the rarest cofactors in biology, F 420 is best known for its role in methanogenesis and has only been chemically identified in two phyla to date, the Euryarchaeota and Actinobacteria. In this work, we show that this cofactor is more widely distributed than previously reported. We detected the genes encoding all five known F 420 biosynthesis enzymes (cofC, cofD, cofE, cofG and cofH) in at least 653 bacterial and 173 archaeal species, including members of the dominant soil phyla Proteobacteria, Chloroflexi and Firmicutes. Metagenome datamining validated that these genes were disproportionately abundant in aerated soils compared with other ecosystems. We confirmed through high-performance liquid chromatography analysis that aerobically grown stationary-phase cultures of three bacterial species, Paracoccus denitrificans, Oligotropha carboxidovorans and Thermomicrobium roseum, synthesized F 420 , with oligoglutamate sidechains of different lengths. To understand the evolution of F 420 biosynthesis, we also analyzed the distribution, phylogeny and genetic organization of the cof genes. Our data suggest that although the F o precursor to F 420 originated in methanogens, F 420 itself was first synthesized in an ancestral actinobacterium. F 420 biosynthesis genes were then disseminated horizontally to archaea and other bacteria. Together, our findings suggest that the cofactor is more significant in aerobic bacterial metabolism and soil ecosystem composition than previously thought. The cofactor may confer several competitive advantages for aerobic soil bacteria by mediating their central metabolic processes and broadening the range of organic compounds they can synthesize, detoxify and mineralize.

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.

F420 is a microbial cofactor that mediates a wide range of physiologically important and industrially relevant redox reactions, including in methanogenesis and tetracycline biosynthesis. This deazaflavin comprises a redox-active isoalloxazine headgroup conjugated to a lactyloligoglutamyl tail. Here we studied the catalytic significance of the oligoglutamate chain, which differs in length between bacteria and archaea. We purified short-chain F420 (two glutamates) from a methanogen isolate and long-chain F420 (five to eight glutamates) from a recombinant mycobacterium, confirming their different chain lengths by HPLC and LC/MS analysis. F420 purified from both sources was catalytically compatible with purified enzymes from the three major bacterial families of F420-dependent oxidoreductases. However, long-chain F420 bound to these enzymes with a six- to ten-fold higher affinity than short-chain F420. The cofactor side chain also significantly modulated the kinetics of the enzymes, with long-chain F420 increasing the substrate affinity (lower Km) but reducing the turnover rate (lower kcat) of the enzymes. Molecular dynamics simulations and comparative structural analysis suggest that the oligoglutamate chain of F420 makes dynamic electrostatic interactions with conserved surface residues of the oxidoreductases while the headgroup binds the catalytic site. In conjunction with the kinetic data, this suggests that electrostatic interactions made by the oligoglutamate tail result in higher-affinity, lower-turnover catalysis. Physiologically, we propose that bacteria have selected for long-chain F420 to better control cellular redox reactions despite tradeoffs in catalytic rate. Conversely, this suggests that industrial use of shorter-length F420 will greatly increase the rates of bioremediation and biocatalysis processes relying on purified F420-dependent oxidoreductases.

SdhE is required for the flavinylation and activation of succinate dehydrogenase and fumarate reductase (FRD). In addition, SdhE is conserved in proteobacteria (α, β and γ) and eukaryotes. Although the function of this recently characterized family of proteins has been determined, almost nothing is known about how their genes are regulated. Here, the RsmA (CsrA) and RsmC (HexY) post-transcriptional and post-translational regulators have been identified and shown to repress sdhEygfX expression in Serratia sp. ATCC 39006. Conversely, the flagella master regulator complex, FlhDC, activated sdhEygfX transcription. To investigate the hierarchy of control, we developed a novel approach that utilized endogenous CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated) genome-editing by a type I-F system to generate a chromosomal point mutation in flhC. Mutation of flhC alleviated the ability of RsmC to repress sdhEygfX expression, whereas RsmA acted in both an FlhDC-dependent and -independent manner to inhibit sdhEygfX. Mutation of rsmA or rsmC, or overexpression of FlhDC, led to increased prodigiosin, biosurfactant, swimming and swarming. Consistent with the modulation of sdhE by motility regulators, we have demonstrated that SdhE and FRD are required for maximal flagella-dependent swimming. Together, these results demonstrate that regulators of both metabolism and motility (RsmA, RsmC and FlhDC) control the transcription of the sdhEygfX operon.

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.

28F420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, 29 this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and 30 activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent 31 biochemical study proposed a revised biosynthesis pathway for F420 in mycobacteria; it was 32 suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather 33 than 2-phospholactate as long proposed, but these findings were subsequently challenged. In 34 this work, we combined metabolomic, genetic, and structural analyses to resolve these 35 discrepancies and determine the basis of F420 biosynthesis in mycobacterial cells. We show that, 36 in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-37 phospholactate stimulates F420 biosynthesis. Analysis of F420 biosynthesis intermediates 38 present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic 39 pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor 40 compound dehydro-F420-0. To determine the structural basis of dehydro-F420-0 production, we 41 solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo, substrate, 42 and product bound forms. These data show the essential role of a single divalent cation in 43 coordinating the catalytic pre-complex of this enzyme and demonstrate that dehydro-F420-0 44 synthesis occurs through a direct substrate transfer mechanism. Together, these findings 45 resolve the biosynthetic pathway of F420 in mycobacteria and have significant implications for 46 understanding the emergence of antitubercular prodrug resistance. 47 48 49 50 51 52 53 54 55 56Factor 420 (F420) is a deazaflavin cofactor that mediates diverse redox reactions in bacteria and 57 archaea (1). Chemically, F420 consists of a redox-active deazaflavin headgroup (derived from 58 the chromophore Fo) that is conjugated to a variable-length polyglutamate tail via a 59 phosphoester linkage (2). While the Fo headgroup of F420 superficially resembles flavins (e.g. 60FAD, FMN), three chemical substitutions in the isoalloxazine ring give it distinct chemical 61 properties more reminiscent of nicotinamides (e.g. NADH, NADPH) (1). These include a low 62 standard potential (-350 mV) and obligate two-electron (hydride) transfer chemistry (3, 4). The 63 electrochemical properties of F420 make it ideal to reduce a wide range of otherwise 64 recalcitrant organic compounds (5-7). Diverse prokaryotes are known to synthesize F420, but 65 the compound is best characterised for its roles in methanogenesis in archaea, antibiotic 66 biosynthesis in streptomycetes, and metabolic adaptation of mycobacteria (1,(8)(9)(10)(11). In 67 mycobacteria, F420 is involved in a plethora of processes: central carbon metabolism, cell wall 68 synthesis, recovery from dormancy, resistance to oxidative stress, and inactivation of certain 69 bactericidal agents (7,(12)(13)(1...