Cofactor F420: an expanded view of its distribution, biosynthesis and roles in bacteria and archaea

Abstract

Many bacteria and archaea produce the redox cofactor F420. F420 is structurally similar to the cofactors FAD and FMN but is catalytically more similar to NAD and NADP. These properties allow F420 to catalyze challenging redox reactions, including key steps in methanogenesis, antibiotic biosynthesis and xenobiotic biodegradation. In the last 5 years, there has been much progress in understanding its distribution, biosynthesis, role and applications. Whereas F420 was previously thought to be confined to Actinobacteria and Euryarchaeota, new evidence indicates it is synthesized across the bacterial and archaeal domains, as a result of extensive horizontal and vertical biosynthetic gene transfer. F420 was thought to be synthesized through one biosynthetic pathway; however, recent advances have revealed variants of this pathway and have resolved their key biosynthetic steps. In parallel, new F420-dependent biosynthetic and metabolic processes have been discovered. These advances have enabled the heterologous production of F420 and identified enantioselective F420H2-dependent reductases for biocatalysis. New research has also helped resolve how microorganisms use F420 to influence human and environmental health, providing opportunities for tuberculosis treatment and methane mitigation. A total of 50 years since its discovery, multiple paradigms associated with F420 have shifted, and new F420-dependent organisms and processes continue to be discovered.

Keywords: cofactor 420; cofactor biosynthesis; cofactor distribution; enzymology; redox chemistry; redox cofactor.

Figure 1.

Structural comparison of F₄₂₀ with flavins and nicotinamides. (A) Structures of the riboflavin head group and tail groups of the flavins FMN and FAD. (B) Structures of the 5-deazaflavin head group and tail groups of F₄₂₀ and 3PG-F₄₂₀. Locations of chemical substitutions between riboflavin and 5-deazaflavin are highlighted. N = 2–9 depending on the microbial species. (C) Structural similarity between the nicotinamides NAD(P)H and the central redox-active portion of F₄₂₀H₂. For F₄₂₀H_2, R represents the phospholactyl and oligoglutamate tail shown in panel B. For NAD(P)H, R2 represents the ribose-5-phosphate of the nicotinamide nucleotide and the adenosine nucleobase as shown in (Bogan and Brenner 2013).

Figure 2.

F₄₂₀ protonaiton states, redox transitions and associated spectral shifts. (A) Changes in the protonation state of F₄₂₀ and F₄₂₀H₂ as a result of the change in external pH. R = F₄₂₀ tail group as depicted in Fig. 1B. (B) Spectral changes of F₄₂₀ between pH 5.5 and 9.0 resulting from a change in protonation state depicted in panel A. Inset graph shows a change in absorbance at 420 nm. (C) Spectral change in F₄₂₀H₂ as in panel B. Inset graph shows changes in absorbance at 280 nm. Panels B and C are adapted from Mohamed et al. (2016a).

Figure 3.

F ₄₂₀-dependent enzyme families that reduce or oxidize substrates via direct hydride transfer. Representative structures are shown of families of F₄₂₀-dependent oxidoreductases in complex with F₄₂₀ and substrate, inhibitor, or substrate analog. Inhibitors or substrate analogs are indicated with *. The secondary structural elements are highlighted (blue = α-helix, yellow = β-sheet or coil). (A) FDOR-A family F₄₂₀H₂-dependent menaquinone reductase (Ddn) from M. tuberculosis docked with menadione (PDB ID = 3R5R; Cellitti et al. 2012). (B) FDOR-B family enzyme of unknown function MSMEG_6526 from M. smegmatis in complex with 2-methyl-2,4-pentanediol (MPD; PDB ID = 4ZKY; Ahmed et al. 2015). (C) Rossmann-A fold enzyme NADPH:F₄₂₀ oxidoreductase (Fno) from A. fulgidus in complex with NADPH (PDB ID = 1JAY; Warkentin et al. 2001). (D) LLHT family F₄₂₀-reducing glucose-6-phosphate dehydrogenase (Fgd) from M. tuberculosis in complex with citrate (PDB ID = 3B4Y; Bashiri et al. 2008). The region of protein capping the active site is rendered transparent for clarity. (E) Rossmann-B fold enzyme F₄₂₀-dependent CH₂=H₄MPT dehydrogenase (Mtd) from Methanopyrus kandleri in complex with CH₂=H₄MPT (PDB ID = 3IQE; Ceh et al. 2009).

Figure 4.

F₄₂₀-dependent enzymes that mediate oxidation or reduction indirectly via flavin. Representative structures or models of families F₄₂₀-dependent oxidoreductases that mediate hydride transfer via a bound flavin cofactor. Structures generated via homology modeling using Phyre2 (Kelley et al. 2015) are indicated with *. The secondary structural elements are highlighted (blue = α-helix, yellow = β-sheet or coil), FMN or FAD colored red and FeS clusters and metal ions are shown as spheres. (A) FDFO family F₄₂₀H₂-dependent flavodiiron oxidase (FprA) from Methanothermobacter thermautotrophicus responsible for the reduction of O₂ to H₂O (PDB ID = 2OHJ; Seedorf et al. 2007). (B) FDRC domain-containing F₄₂₀-reducing NiFe hydrogenase (Frh) from Methanothermobacter marburgensis (PDB ID = 4CI0; Allegretti et al. 2014). (C) F₄₂₀H₂-dependent thioredoxin reductase (DFTR) from M. jannaschii (homology model; Susanti, Loganathan and Mukhopadhyay 2016).

Figure 5.

Phylogenetic distribution of F_O and F₄₂₀ producing organisms. A simplified two-domain tree of life depicted the organisms shown or predicted to produce the 5-deazaflavins F_O or F₄₂₀. This is based on currently available data from published work (Greening et al. ; Ney et al. 2017a), and genomic and metagenomic data in the NCBI database (as of October 2020). Tree topography is based on Hug et. al. (Hug et al. 2016) and Castelle and Banfield (2018), with additional reference to Zhou et al. (2020), Wang et al. (2019) and Momper, Aronson and Amend (2018). * = F₄₂₀ biosynthesis genes detected only in multiple metagenome-assembled genomes (MAGs) or single-amplified genomes (SAGs) from these archaea and bacteria, rather than genomes derived from pure culture.

Figure 6.

F₄₂₀-dependent reactions of one-carbon metabolism in archaea. F₄₂₀ is a cofactor involved in key steps in hydrogenotrophic methanogenesis, methylotrophic methanogenesis, anaerobic methanotrophy and anaerobic alkane oxidation in archaea. Hydride transfer reactions involving F₄₂₀-dependent enzymes are indicated as is the enzyme responsible. F₄₂₀H₂ reduced through the oxidation of formate (Ffd), H₂ (Frh), or secondary alcohols (Adf) can be utilized for reactions mediated by Mtd, Mer, or for other cellular processes. Only reactions mediated by F₄₂₀-dependent enzymes are shown in detail. For a full outline of methanogenesis pathways, refer to the following reviews on the subject (Deppenmeier ; Thauer et al. ; Timmers et al. ; Evans et al. 2019).

Figure 7.

Physiological reactions proposed to be mediated by F₄₂₀-dependent enzymes in mycobacteria. The bond oxidized or reduced is highlighted in orange for each substrate, with the enzyme responsible for the reaction indicated. For the reactions shown in A, B and D, F₄₂₀H₂ is generated by Fgd through oxidation of G6P. For the reaction shown in C, F₄₂₀ oxidizes hydroxymycolic acid to ketomycolic acid at the extracellular face of the cytoplasmic membrane, yielding F₄₂₀H₂.

Figure 8.

Reactions proposed to be mediated by F₄₂₀-dependent reductases in streptomycetes. The bond reduced is highlighted in orange for each substrate, with the enzyme responsible for the reaction indicated. F₄₂₀H₂ for the reactions shown is generated by the enzyme Fno via the oxidation of NADPH.

Figure 9.

Diverse routes to F₄₂₀ biosynthesis employed by bacteria and archaea. 1 = classical archaeal pathway (Euryarchaeota), 2 = bacterial pathway a (Actinobacteria, Chloroflexi), 3 = bacterial pathway b (Betaproteobacteria). The substrates and mechanisms for F_O biosynthesis are shared between all identified pathways. Abbreviated compounds are as follows: PEP, phosphoenolpyruvate; 2PL, 2-phospho-L-lactate; 3PG, 3-phosphoglycerate; EPPG, enolpyruvyl-diphospho-5’-guanosine; LPPG, lactyl-diphospho-5’-guanosine; GPPG, 3-guanosine-5’-disphospho-D-glycerate. The enzymes involved in each biosynthesis step are indicated.

Figure 10.

Genetic organization of F₄₂₀ biosynthetic genes in bacteria and archaea. A schematic of the generalized the genetic context of F₄₂₀ biosynthetic genes in experimentally confirmed (panel A) and predicted (panel B) F₄₂₀-producing bacteria (left) and archaea (right). F₄₂₀ biosynthetic genes are labeled and color-coded. Additional F₄₂₀ related genes are colored grey and labeled as follows: Fgd, F₄₂₀-reducing glucose 6-phosphate dehydrogenase; Fno, F₄₂₀-reducing NADPH dehydrogenase; FDOR, predicted F₄₂₀H₂-dependent reductase; LLHT, predicted F₄₂₀H₂-dependent luciferase-like hydride transferase; Mer, F₄₂₀H₂-dependent CH₂=H₄MPT reductase. Hypothetical genes or those with no known F₄₂₀-related function are shown and colored white. Black tilde symbols designate undefined intergenomic space. Gene context is adapted from Ney et al. (2017a) or determined directly from available genome sequences.

Figure 11.

Production of the deazaflavin F_O is catalyzed by dual SAM-radical domains. (A) Structural model of the SAM radical domains that mediate Fo synthesis, consisting of the two separate proteins CofG and CofH (in archaea and some bacteria) or a single fusion protein FbiC (in bacteria and eukaryotes). Structural models constructed based on homology modeling using Phyre2 based on the structure of MqnE from Pedobacter heparinus (PDB ID = 6XI9; Kelley et al. 2015). (B) A summary of the proposed reaction performed by CofH. (C) A summary of the proposed reaction performed by CofG. For the full reaction scheme summarized in panels B and C refer to Philmus et al. (2015). R = The F_O ribose tail as shown in Fig. 9.

Figure 12.

Crystal structure of FbiD from M. tuberculosis in complex with PEP substrate. (A) Cartoon view of the crystal structure of FbiD in complex with PEP (PDB ID = 6BWH). (B) A surface model of the FbiB active site showing the region predicted to bind GTP adopts an occluded conformation. (C) Key residues of FbiD involved in the coordination of the PEP substrate and catalytic Mg²⁺ ions. Atomic distances less than 3.2 Å are shown as dashed yellow lines.

Figure 13.

Crystal structure of the FbiA-substrate complex from M. smegmatis. (A) Crystal structure of the FbiA dimer [PDB ID = 6UW5] in complex with GDP in Mol. A and Fo and EPPG (modeled in place of co-crystallized GDP) in Mol. B. FbiA is shown as a cartoon model, substrate molecules are shown as sticks and a Ca²⁺ ion (likely Mg²⁺ in the active enzyme) is shown as a sphere. (B) A cross-eye stereo view of the active site of FbiA Mol. B from panel A, showing key residues for coordinating the FbiA substrate complex as sticks and coordination distances as dotted lines. (C) A proposed reaction mechanism for synthesis DH-F₄₂₀-0 by FbiA, in which the carboxyl group of EPPG donates an electron to F_O, activating it to perform nucleophilic attack on the EPPG β-phosphate.

Figure 14.

Crystal structure of CofE from A. fulgidus in complex with GDP and Mn²⁺. (A) Cartoon view of the crystal structure of the functional dimer of CofE [PDB ID = 2PHN]. CofE subunits are shown in pink (Mol. A) and red (Mol. B). Bound GDP and catalytic Mn²⁺ ions are shown for Mol. B only, as stick and sphere representation respectively. (B) Electrostatic surface view of the CofE active site showing bound GDP, catalytic Mn²⁺ ions and predicted F₄₂₀-0 binding pocket. (C) Proposed catalytic mechanism for the first γ-linked glutamate addition mediated by CofE, R = F_O minus terminal hydroxyl.

Figure 15.

Crystal structure of FbiB_C-term from M. tuberculosis. (A) Cartoon view of the crystal structure of the functional dimer of the FbiB C-terminal domain responsible for the FMNH₂ mediated reduction of DH-F₄₂₀-0 [PDB IDs = 4XOO, 4XOQ]. FbiB_C-term is shown in green and FMN and DH-_F420-0 (modeled based on the co-crystal structure of F_O) are shown as sticks. (B) A zoomed view of the FbiB_C-term active site in complex with FMN and DH-F₄₂₀-0 as in panel A, with a cartoon and transparent atomic surface of the FbiB_C-term shown.

Figure 16.

Schematic showing possible events in the evolution of F₄₂₀ and its acquisition by different bacterial and archaeal lineages. Arrows with solid lines indicate potential horizontal transfer of ancestral F₄₂₀ biosynthetic genes. Dashed lines with bidirectional arrows indicate a likely gene transfer event of unknown directionality. Phyla labels are simplified for clarity. Note the figure is a speculative model drawn based on data from sources discussed in the main text and other models are also consistent with these data.

Figure 17.

Compounds reduced by purified F₄₂₀-dependent enzymes. (A) Classes of compounds shown to be reduced by purified F₄₂₀-dependent enzymes. The reduced bond is highlighted in orange for each compound. The enzymes tested, as well as their substrate range and relative activity levels, are provided in Table 5. (B) Proposed reaction schemes for the reduction of menaquinone and malachite green by F₄₂₀H₂-dependent reductases of the FDOR-A superfamily. In both cases, initial hydride transfer to one carbon atom of an activated alkene is followed by tautomerization yielding the final reaction product.

Figure 18.

Nitroimidazole prodrugs effective against M. tuberculosis and their activation by Ddn. (A) Structure of nitroimidazole-containing prodrugs developed for tuberculosis treatment. Delamanid and pretomanid were recently approved for the treatment of M. tuberculosis infection, while CGI-17341 was abandoned due to toxicity concerns. The nitroimidazole functional group is highlighted in orange. (B) The complex between pretomanid and its activating enzyme Ddn from M. tuberculosiswas generated by molecular docking using AutoDock Vina (Trott and Olson 2010). The proximity between the nitroimidazole group of pretomanid and the hydride transferring C5 carbon of F₄₂₀ is shown in the inset panel. (C) Proposed products for the breakdown of pretomanid following reduction by Ddn, full reaction schemes leading to product generation refer to Singh et al. (2008).