Cofactor F420, an emerging redox power in biosynthesis of secondary metabolites.

Cofactor F420 is a low-potential hydride-transfer deazaflavin that mediates important oxidoreductive reactions in the primary metabolism of archaea and a wide range of bacteria. Over the past decade, biochemical studies have demonstrated another essential role for F420 in the biosynthesis of various classes of natural products. These studies have substantiated reports predating the structural determination of F420 that suggested a potential role for F420 in the biosynthesis of several antibiotics produced by Streptomyces. In this article, we focus on this exciting and emerging role of F420 in catalyzing the oxidoreductive transformation of various imine, ketone and enoate moieties in secondary metabolites. Given the extensive and increasing availability of genomic and metagenomic data, these F420-dependent transformations may lead to the discovery of novel secondary metabolites, providing an invaluable and untapped resource in various biotechnological applications.

Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis.

The nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10-5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

Elongation of the Poly-γ-glutamate Tail of F420 Requires Both Domains of the F420:γ-Glutamyl Ligase (FbiB) of Mycobacterium tuberculosis.

Cofactor F420is an electron carrier with a major role in the oxidoreductive reactions ofMycobacterium tuberculosis, the causative agent of tuberculosis. A γ-glutamyl ligase catalyzes the final steps of the F420biosynthesis pathway by successive additions ofl-glutamate residues to F420-0, producing a poly-γ-glutamate tail. The enzyme responsible for this reaction in archaea (CofE) comprises a single domain and produces F420-2 as the major species. The homologousM. tuberculosisenzyme, FbiB, is a two-domain protein and produces F420with predominantly 5-7l-glutamate residues in the poly-γ-glutamate tail. The N-terminal domain of FbiB is homologous to CofE with an annotated γ-glutamyl ligase activity, whereas the C-terminal domain has sequence similarity to an FMN-dependent family of nitroreductase enzymes. Here we demonstrate that full-length FbiB adds multiplel-glutamate residues to F420-0in vitroto produce F420-5 after 24 h; communication between the two domains is critical for full γ-glutamyl ligase activity. We also present crystal structures of the C-terminal domain of FbiB in apo-, F420-0-, and FMN-bound states, displaying distinct sites for F420-0 and FMN ligands that partially overlap. Finally, we discuss the features of a full-length structural model produced by small angle x-ray scattering and its implications for the role of N- and C-terminal domains in catalysis.

F420-dependent transformations in biosynthesis of secondary metabolites.

Cofactor F420 has been historically known as the "methanogenic redox cofactor". It is now recognised that F420 has essential roles in the primary and secondary metabolism of archaea and bacteria. Recent discoveries highlight the role of F420 as a redox cofactor in the biosynthesis of various natural products, including ribosomally synthesised and post-translationally modified peptides, and a new class of nicotinamide adenine dinucleotide-based secondary metabolites. With the vast availability of (meta)genomic data, the identification of uncharacterised F420-dependent enzymes offers the potential for discovering novel secondary metabolites, presenting valuable prospects for clinical and biotechnological applications.