Structure of an amide bond forming F(420):gamma-glutamyl ligase from Archaeoglobus fulgidus -- a member of a new family of non-ribosomal peptide synthases.
ABSTRACT: F(420) is a flavin-like redox-active coenzyme commonly used by archaea and some eubacteria in a variety of biochemical reactions in methanogenesis, the formation of secondary metabolites, the degradation of nitroaromatic compounds, activation of nitroimidazofurans, and F(420)-dependent photolysis in DNA repair. Coenzyme F(420)-2 biosynthesis from 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo) and lactaldehyde involves six enzymatic steps and five proteins (CofA, CofB, CofC, CofD, and CofE). CofE, a F(420)-0:gamma-glutamyl ligase, is responsible for the last two enzymatic steps; it catalyses the GTP-dependent addition of two L-glutamate residues to F(420)-0 to form F(420)-2. CofE is found in archaea, the aerobic actinomycetes, and cyanobacteria. Here, we report the first crystal structure of the apo-F(420)-0:gamma-glutamyl ligase (CofE-AF) from Archaeoglobus fulgidus and its complex with GDP at 2.5 A and 1.35 A resolution, respectively. The structure of CofE-AF reveals a novel protein fold with an intertwined, butterfly-like dimer formed by two-domain monomers. GDP and Mn(2+) are bound within the putative active site in a large groove at the dimer interface. We show that the enzyme adds a glutamate residue to both F(420)-0 and F(420)-1 in two distinct steps. CofE represents the first member of a new structural family of non-ribosomal peptide synthases.
Project description:Coenzyme F(420), a hydride carrier, is found in Archaea and some bacteria and has crucial roles in methanogenesis, antibiotic biosynthesis, DNA repair, and activation of antitubercular compounds. CofD, 2-phospho-l-lactate transferase, catalyzes the last step in the biosynthesis of F(420)-0 (F(420) without polyglutamate), by transferring the lactyl phosphate moiety of lactyl(2)diphospho-(5')guanosine to 7,8-didemethyl-8-hydroxy-5-deazariboflavin ribitol (Fo). CofD is highly conserved among F(420)-producing organisms, and weak sequence homologs are also found in non-F(420)-producing organisms. This superfamily does not share any recognizable sequence conservation with other proteins. Here we report the first crystal structures of CofD, the free enzyme and two ternary complexes, with Fo and P(i) or with Fo and GDP, from Methanosarcina mazei. The active site is located at the C-terminal end of a Rossmann fold core, and three large insertions make significant contributions to the active site and dimer formation. The observed binding modes of Fo and GDP can explain known biochemical properties of CofD and are also supported by our binding assays. The structures provide significant molecular insights into the biosynthesis of the F(420) coenzyme. Large structural differences in the active site region of the non-F(420)-producing CofD homologs suggest that they catalyze a different biochemical reaction.
Project description: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.
Project description:In methanogenic Archaea, the final step of methanogenesis generates methane and a heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB). Reduction of this heterodisulfide by heterodisulfide reductase to regenerate HS-CoM and HS-CoB is an exergonic process. Thauer et al. [Thauer, et al. 2008 Nat Rev Microbiol 6:579-591] recently suggested that in hydrogenotrophic methanogens the energy of heterodisulfide reduction powers the most endergonic reaction in the pathway, catalyzed by the formylmethanofuran dehydrogenase, via flavin-based electron bifurcation. Here we present evidence that these two steps in methanogenesis are physically linked. We identify a protein complex from the hydrogenotrophic methanogen, Methanococcus maripaludis, that contains heterodisulfide reductase, formylmethanofuran dehydrogenase, F(420)-nonreducing hydrogenase, and formate dehydrogenase. In addition to establishing a physical basis for the electron-bifurcation model of energy conservation, the composition of the complex also suggests that either H(2) or formate (two alternative electron donors for methanogenesis) can donate electrons to the heterodisulfide-H(2) via F(420)-nonreducing hydrogenase or formate via formate dehydrogenase. Electron flow from formate to the heterodisulfide rather than the use of H(2) as an intermediate represents a previously unknown path of electron flow in methanogenesis. We further tested whether this path occurs by constructing a mutant lacking F(420)-nonreducing hydrogenase. The mutant displayed growth equal to wild-type with formate but markedly slower growth with hydrogen. The results support the model of electron bifurcation and suggest that formate, like H(2), is closely integrated into the methanogenic pathway.
Project description:Archae possess unique biochemical systems quite distinct from the pathways present in eukaryotes and eubacteria. 7,8-Dimethyl-8-hydroxy-5deazaflavin (F(0)) and F(420) are unique deazaflavin-containing coenzyme and methanogenic signature molecules, essential for a variety of biochemical transformations associated with methane biosynthesis and light-dependent DNA repair. The deazaflavin cofactor system functions during methane biosynthesis as a low-potential hydrid shuttle F(420)/F(420)H(2). In DNA photolyase repair proteins, the deazaflavin cofactor is in the deprotonated state active as a light-collecting energy transfer pigment. As such, it converts blue sunlight into energy used by the proteins to drive an essential repair process. Analysis of a eukaryotic (6-4) DNA photolyase from Drosophila melanogaster revealed a binding pocket, which tightly binds F(0). Residues in the pocket activate the cofactor by deprotonation so that light absorption and energy transfer are switched on. The crystal structure of F(0) in complex with the D. melanogaster protein shows the atomic details of F(0) binding and activation, allowing characterization of the residues involved in F(0) activation. The results show that the F(0)/F(420) coenzyme system, so far believed to be strictly limited to the archael kingdom of life, is far more widespread than anticipated. Analysis of a D. melanogaster extract and of a DNA photolyase from the primitive eukaryote Ostreococcus tauri provided direct proof for the presence of the F(0) cofactor also in higher eukaryotes.
Project description:Methylenetetratetrahydromethanopterin reductase (Mer) is involved in CO(2) reduction to methane in methanogenic archaea and catalyses the reversible reduction of methylenetetrahydromethanopterin (methylene-H(4)MPT) to methyl-H(4)MPT with coenzyme F(420)H(2), which is a reduced 5'-deazaflavin. Mer was recently established as a TIM barrel structure containing a nonprolyl cis-peptide bond but the binding site of the substrates remained elusive. We report here on the crystal structure of Mer in complex with F(420) at 2.6 A resolution. The isoalloxazine ring is present in a pronounced butterfly conformation, being induced from the Re-face of F(420) by a bulge that contains the non-prolyl cis-peptide bond. The bindingmode of F(420) is very similar to that in F(420)-dependent alcohol dehydrogenase Adf despite the low sequence identity of 21%. Moreover, binding of F(420) to the apoenzyme was only associated with minor conformational changes of the polypeptide chain. These findings allowed us to build an improved model of FMN into its binding site in bacterial luciferase, which belongs to the same structural family as Mer and Adf and also contains a nonprolyl cis-peptide bond in an equivalent position.
Project description:The use of molecular hydrogen as electron donor for energy generation is a defining characteristic of the hydrogenotrophic methanogens, an ancient group that dominates the phylum Euryarchaeota. We present here a global study of changes in mRNA abundance in response to hydrogen availability for a hydrogenotrophic methanogen. Cells of Methanococcus maripaludis were grown by using continuous culture to deconvolute the effects of hydrogen limitation and growth rate, and microarray analyses were conducted. Hydrogen limitation markedly increased mRNA levels for genes encoding enzymes of the methanogenic pathway that reduce or oxidize the electron-carrying deazaflavin, coenzyme F(420). F(420)-dependent redox functions in energy-generating metabolism are characteristic of the methanogenic Archaea, and the results show that their regulation is distinct from other redox processes in the cell. Rapid growth increased mRNA levels of the gene for an unusual hydrogenase, the hydrogen-dependent methylenetetrahydromethanopterin dehydrogenase.
Project description:A metaproteomic approach was used to analyse the proteins expressed and provide functional evidence of key metabolic pathways in the combined production of hydrogen and methane by anaerobic fermentation (CHMP-AF) for reed straw utilisation. The functions and structures of bacteria and archaea populations show significant succession in the CHMP-AF process. There are many kinds of bacterial functional proteins, mainly belonging to phyla Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes, that are involved in carbohydrate metabolism, energy metabolism, lipid metabolism, and amino acid metabolism. Ferredoxin-NADP reductase, present in bacteria in genus Azotobacter, is an important enzyme for NADH/NAD+ equilibrium regulation in hydrogen production. The archaeal functional proteins are mainly involved in methane metabolism in energy metabolism, such as acetyl-CoA decarboxylase, and methyl-coenzyme M reductase, and the acetic acid pathway exhibited the highest proportion of the total. The archaea of genus Methanosarcina in phylum Euryarchaeota can produce methane under the effect of multi-functional proteins through acetic acid, CO2 reduction, and methyl nutrient pathways. The study demonstrates metaproteomics as a new way of uncovering community functional and metabolic activity. The combined information was used to identify the metabolic pathways and organisms crucial for lignocellulosic biomass degradation and biogas production. This also regulates the process from its protein levels and improves the efficiency of biogas production using reed straw biomass.
Project description:The biosynthetic precursor of redox cofactor F420, 7,8-didemethyl-8-hydroxy-5-deazariboflavin, was prepared in four steps from 6-chlorouracil, 2-chloro-4-hydroxybenzaldehyde and bis-isopropylidene protected D-ribose. The latter aldehyde was transformed to the corresponding protected ribitylamine via the oxime, which was submitted to reduction with LiAlH4. Key advantage compared to previous syntheses is the utilization of a polyol-protective group which allowed the chromatographic purification of a key-intermediate product providing the target compound with high purity.
Project description:To avoid the dangerous operational conditions, shorten the preparation time, and improve the adsorption performance of amino-functionalized nanomagnetic materials with a core?shell structure, a magnetic nanocomposite of CoFe?O?@SiO? was successfully functionalized with amino group (-NH?) through a mild and facile hydrothermal method without the use of any toxic or harmful solvents at a relatively low temperature. The preparation time of the key steps of amino functionalization was shortened from 30 h to about 10 h. The core-shell structure and successful grafting were confirmed by various means. The amino-functionalized CoFe?O?@SiO? was used for the removal mercury (Hg(II)), a heavy metal, and exhibited excellent magnetic properties and a high Langmuir adsorption capacity of 149.3 mg Hg(II)/g. The adsorption of Hg(II) onto CoFe?O?@SiO??NH? followed the pseudo-second-order kinetic equation and Langmuir model. The thermodynamic data showed that the Hg(II) adsorption process was achieved through spontaneous exothermic and monolayer adsorption with electrostatic adsorption and chemisorption. In addition, the as-prepared CoFe?O?@SiO??NH? nanoparticles had a good reusable value, good application performance and stability, and can provide a mild and facile way to remove heavy metals from aqueous solution.
Project description: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 ?-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 ?-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.