Cofactor F₄₂₀ 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 F₄₂₀ 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-F₄₂₀-0. The C-terminal domain of FbiB then utilizes FMNH₂ to reduce dehydro-F₄₂₀-0, which produces mature F₄₂₀ species when combined with the γ-glutamyl ligase activity of the N-terminal domain. These new insights have allowed the heterologous production of F₄₂₀ from a recombinant F₄₂₀ biosynthetic pathway in Escherichia coli.

The asymmetric reduction of enoates, imines and ketones are among the most important reactions in biocatalysis. These reactions are routinely conducted using enzymes that use nicotinamide cofactors as reductants. The deazaflavin cofactor F₄₂₀ also has electrochemical properties that make it suitable as an alternative to nicotinamide cofactors for use in asymmetric reduction reactions. However, cofactor F₄₂₀-dependent enzymes remain under-explored as a resource for biocatalysis. This review considers the cofactor F₄₂₀-dependent enzyme families with the greatest potential for the discovery of new biocatalysts: the flavin/deazaflavin-dependent oxidoreductases (FDORs) and the luciferase-like hydride transferases (LLHTs). The characterized F₄₂₀-dependent reductions that have the potential for adaptation for biocatalysis are discussed, and the enzymes best suited for use in the reduction of oxidized cofactor F₄₂₀ to allow cofactor recycling in situ are considered. Further discussed are the recent advances in the production of cofactor F₄₂₀ and its functional analog F_O-5′-phosphate, which remains an impediment to the adoption of this family of enzymes for industrial biocatalytic processes. Finally, the prospects for the use of this cofactor and dependent enzymes as a resource for industrial biocatalysis are discussed.

The ability to persist in the absence of growth triggered by low-oxygen levels is a critical process for the survival of mycobacterial species in many environmental niches. MSMEG_5243 (fsq), a gene of unknown function in Mycobacterium smegmatis, is up-regulated in response to hypoxia and regulated by DosRDosS/DosT, an oxygen- and redox-sensing two-component system that is highly conserved in mycobacteria. In this communication, we demonstrate that MSMEG_5243 is a flavin-sequestering protein and henceforth refer to it as Fsq. Using an array of biochemical and structural analyses, we show that Fsq is a member of the diverse superfamily of flavin- and deazaflavin-dependent oxidoreductases (FDORs) and is widely distributed in mycobacterial species. We created a markerless deletion mutant of fsq and demonstrate that fsqis required for cell survival during hypoxia. Using fsq deletion and overexpression, we found that fsq enhances cellular resistance to hydrogen peroxide treatment. The X-ray crystal structure of Fsq, solved to 2.7 Å, revealed a homodimeric organization with FAD bound noncovalently. The Fsq structure also uncovered no potential substrate-binding cavities, as the FAD is fully enclosed, and electrochemical studies indicated that the Fsq:FAD complex is relatively inert and does not share common properties with electron-transfer proteins. Taken together, our results suggest that Fsq reduces the formation of reactive oxygen species (ROS) by sequestering free FAD during recovery from hypoxia, thereby protecting the cofactor from undergoing autoxidation to produce ROS. This finding represents a new paradigm in mycobacterial adaptation to hypoxia.

The deazaflavin cofactor F₄₂₀ is a low-potential, two-electron redox cofactor produced by some Archaea and Eubacteria that is involved in methanogenesis and methanotrophy, antibiotic biosynthesis, and xenobiotic metabolism. However, it is not produced by bacterial strains commonly used for industrial biocatalysis or recombinant protein production, such as Escherichia coli, limiting our ability to exploit it as an enzymatic cofactor and produce it in high yield. Here we have utilized a genome-scale metabolic model of E. coli and constraint-based metabolic modelling of cofactor F₄₂₀ biosynthesis to optimize F₄₂₀ production in E. coli. This analysis identified phospho-enol pyruvate (PEP) as a limiting precursor for F₄₂₀ biosynthesis, explaining carbon source-dependent differences in productivity. PEP availability was improved by using gluconeogenic carbon sources and overexpression of PEP synthase. By improving PEP availability, we were able to achieve a ~ 40-fold increase in the space–time yield of F₄₂₀ compared with the widely used recombinant Mycobacterium smegmatis expression system. This study establishes E. coli as an industrial F₄₂₀-production system and will allow the recombinant in vivo use of F₄₂₀-dependent enzymes for biocatalysis and protein engineering applications.

Asymmetric reduction by ene‐reductases has received considerable attention in recent decades. While several enzyme families possess ene‐reductase activity, the Old Yellow Enzyme (OYE) family has received the most scientific and industrial attention. However, there is a limited substrate range and few stereocomplementary pairs of current ene‐reductases, necessitating the development of a complementary class. Flavin/deazaflavin oxidoreductases (FDORs) that use the uncommon cofactor F₄₂₀ have recently gained attention as ene‐reductases for use in biocatalysis due to their stereocomplementarity with OYEs. Although the enzymes of the FDOR‐As sub‐group have been characterized in this context and reported to catalyse ene‐reductions enantioselectively, enzymes from the similarly large, but more diverse, FDOR‐B sub‐group have not been investigated in this context. In this study, we investigated the activity of eight FDOR‐B enzymes distributed across this sub‐group, evaluating their specific activity, kinetic properties, and stereoselectivity against α,β‐unsaturated compounds. The stereochemical outcomes of the FDOR‐Bs are compared with enzymes of the FDOR‐A sub‐group and OYE family. Computational modelling and induced‐fit docking are used to rationalize the observed catalytic behaviour and proposed a catalytic mechanism.

Protein-protein interactions (PPIs) mediate many fundamental cellular processes and their control through optically or chemically responsive protein domains has a profound impact on basic research and some clinical applications. Most available chemogenetic methods induce the association, i.e., dimerization or oligomerization, of target proteins, and the few available dissociation approaches either break large oligomeric protein clusters or heteromeric complexes. Here, we have exploited the controlled dissociation of a dimeric oxidoreductase from mycobacteria (MSMEG_2027) by its native cofactor, F ₄₂₀ , which is not present in mammals, as a bioorthogonal monomerization switch. We found that in the absence of F ₄₂₀ , MSMEG_2027 forms a unique domain-swapped dimer that occludes the cofactor binding site. Substantial remodelling of the intertwined N-terminal helix upon F ₄₂₀ binding results in the dissolution of the dimer. We then show that MSMEG_2027 can be expressed as fusion proteins in human cells and apply it as a tool to induce and release MAPK/ERK signalling downstream of a chimeric fibroblast growth factor receptor 1 (FGFR1) tyrosine kinase. This F ₄₂₀ -dependent chemogenetic de-dimerization tool is stoichiometric, based on a single domain and presents a novel mechanism to investigate protein complexes in situ .