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Rudolf K Thauer - One of the best experts on this subject based on the ideXlab platform.

  • Si-face stereospecificity at C5 of Coenzyme F420 for F420H2 oxidase from methanogenic Archaea as determined by mass spectrometry.
    The FEBS journal, 2005
    Co-Authors: Henning Seedorf, Jörg Kahnt, Antonio J. Pierik, Rudolf K Thauer
    Abstract:

    Coenzyme F420 is a 5-deazaflavin. Upon reduction, 1,5 dihydro-Coenzyme F420 is formed with a prochiral centre at C5. All the Coenzyme F420-dependent enzymes investigated to date have been shown to be Si-face stereospecific with respect to C5 of the deazaflavin, despite most F420-dependent enzymes being unrelated phylogenetically. In this study, we report that the recently discovered F420H2 oxidase from methanogenic Archaea is also Si-face stereospecific. The enzyme was found to catalyse the oxidation of (5S)-[5-2H1]F420H2 with O2 to [5-1H]F420 rather than to [5-2H]F420 as determined by MALDI-TOF MS. (5S)-[5-2H1]F420H2 was generated by stereospecific enzymatic reduction of F420 with (14a-2H2)-[14a-2H2] methylenetetrahydromethanopterin.

  • F420H2 oxidase (FprA) from Methanobrevibacter arboriphilus, a Coenzyme F420-dependent enzyme involved in O2 detoxification
    Archives of Microbiology, 2004
    Co-Authors: Henning Seedorf, Annette Dreisbach, Reiner Hedderich, Seigo Shima, Rudolf K Thauer
    Abstract:

    Cell suspensions of Methanobrevibacter arboriphilus catalyzed the reduction of O2 with H2 at a maximal specific rate of 0.4 U (μmol/min) per mg protein with an apparent Km for O2 of 30 μM. The reaction was not inhibited by cyanide. The oxidase activity was traced back to a Coenzyme F420-dependent enzyme that was purified to apparent homogeneity and that catalyzed the oxidation of 2 F420H2 with 1 O2 to 2 F420 and 2 H2O. The apparent Km for F420 was 30 μM and that for O2 was 2 μM with a Vmax of 240 U/mg at 37°C and pH 7.6, the pH optimum of the oxidase. The enzyme did not use NADH or NADPH as electron donor or H2O2 as electron acceptor and was not inhibited by cyanide. The 45-kDa protein, whose gene was cloned and sequenced, contained 1 FMN per mol and harbored a binuclear iron center as indicated by the sequence motif H–X–E–X–D–X62–H–X18–D–X60–H. Sequence comparisons revealed that the F420H2 oxidase from M. arboriphilus is phylogenetically closely related to FprA from Methanothermobacter marburgensis (71% sequence identity), a 45-kDa flavoprotein of hitherto unknown function, and to A-type flavoproteins from bacteria (30–40%), which all have dioxygen reductase activity. With heterologously produced FprA from M. marburgensis it is shown that this protein is also a highly efficient F420H2 oxidase and that it contains 1 FMN and 2 iron atoms. The presence of F420H2 oxidase in methanogenic archaea may explain why some methanogens, e.g., the Methanobrevibacter species in the termite hindgut, cannot only tolerate but thrive under microoxic conditions.

  • function of h2 forming methylenetetrahydromethanopterin dehydrogenase from methanobacterium thermoautotrophicum in Coenzyme F420 reduction with h2
    Archives of Microbiology, 1998
    Co-Authors: C Afting, Andreas Hochheimer, Rudolf K Thauer
    Abstract:

    In most methanogenic archaea, two hydrogenase systems that can catalyze the reduction of Coenzyme F420 (F420) with H2 are present: (1) the F420-reducing hydrogenase, which is a nickel iron-sulfur flavoprotein composed of three different subunits, and (2) the N 5, N10-methylenetetrahydromethanopterin dehydrogenase system, which is composed of H2-forming methylenetetrahydromethanopterin dehydrogenase and F420-dependent methylenetetrahydromethanopterin dehydrogenase, both metal-free proteins without an apparent prosthetic group. We report here that in nickel-limited chemostat cultures of Methanobacterium thermoautotrophicum, the specific activity of the F420-reducing Ni/Fe-hydrogenase was essentially zero, whereas that of the H2-forming methylenetetrahydromethanopterin dehydrogenase was six times higher, and that of the F420-dependent methylenetetrahydromethanopterin dehydrogenase was four times higher than in cells grown under non-nickel-limited conditions. This evidence supports the hypothesis that when M. thermoautotrophicum grows under conditions of nickel limitation, the reduction of F420 with H2 is catalyzed by the metal-free methylenetetrahydromethanopterin dehydrogenase system.

  • function of Coenzyme F420 dependent nadp reductase in methanogenic archaea containing an nadp dependent alcohol dehydrogenase
    Archives of Microbiology, 1997
    Co-Authors: Holger Berk, Rudolf K Thauer
    Abstract:

    Methanogenic archaea growing on ethanol or isopropanol as the electron donor for CO2 reduction to CH4 contain either an NADP-dependent or a Coenzyme F420-dependent alcohol dehydrogenase. We report here that in both groups of methanogens, the N5, N10-methylenetetrahydromethanopterin dehydrogenase and the N5, N10-methylenetetrahydromethanopterin reductase, two enzymes involved in CO2 reduction to CH4, are specific for F420. This raised the question how F420H2 is regenerated in the methanogens with an NADP-dependent alcohol dehydrogenase. We found that these organisms contain catabolic activities of an enzyme catalyzing the reduction of F420 with NADPH. The F420-dependent NADP reductase from Methanogenium organophilum was purified and characterized. The N-terminal amino acid sequence showed 42% sequence identity to a putative gene product in Methanococcus jannaschii, the total genome of which has recently been sequenced.

  • Overexpression of the Coenzyme‐F420‐Dependent N5,N10‐Methylenetetrahydromethanopterin Dehydrogenase Gene from the Hyperthermophilic Methanopyrus Kandleri
    European journal of biochemistry, 1997
    Co-Authors: Andreas R Klein, Rudolf K Thauer
    Abstract:

    The mtd gene encoding Coenzyme-F420-dependent N5,N10-methylenetetrahydromethanopterin dehydrogenase (Mtd) in the hyperthermophilic Methanopyrus kandleri has been cloned, sequenced and functionally overexpressed in Escherichia coli. The overproduced enzyme was purified in a 90% yield to apparent homogeneity by means of only one chromatographic step. Its thermostability properties and most of its catalytic properties were the same as those of the native enzyme purified directly from M. kandleri. Only the dependence of the activity on the concentration of lyotropic salts differed slightly. Northern blot analysis revealed that in M. kandleri the mtd gene is monocistronically transcribed.

Andreas R Klein - One of the best experts on this subject based on the ideXlab platform.

Lacy Daniels - One of the best experts on this subject based on the ideXlab platform.

  • Demonstration that fbiC Is Required by Mycobacterium bovis BCG for Coenzyme F420 and FO Biosynthesis
    Journal of bacteriology, 2002
    Co-Authors: Kwang-pil Choi, Nathan Kendrick, Lacy Daniels
    Abstract:

    The structure of Coenzyme F420 (a 7,8-didemethyl-8-hydroxy 5-deazaflavin electron transfer agent that is present in few microorganisms) was first determined in studies with the methanogenic Archaea (13, 14). This Coenzyme had been previously purified from a methanogen, and its spectral and fluorescence properties were described in detail by Cheeseman et al. (6), who gave it its name (for a factor that absorbed maximally in visible wavelengths at 420 nm). However, an earlier report by Cousins described a yellow compound of unknown structure from Mycobacterium smegmatis that, based on its UV-visible spectrum, was almost certainly F420 (11). Naraoka et al. reported that F420 was present in Mycobacterium avium (37), and it was subsequently discovered that F420 was present in Mycobacterium tuberculosis (12) and all other mycobacteria examined (2, 43). Methanobacterium thermoautotrophicum F420 contains two glutamyl residues (14), but Mycobacterium species contain primarily five- and six-glutamyl forms of F420 (F420-5 and F420-6) (2). The structure of F420-5 is shown in Fig. ​Fig.1A1A. FIG. 1. Structure of F420-5 and an overview of the hypothetical pathway for F420 biosynthesis. (A) F420-5 (2); (B) overview of the hypothetical pathway for F420 biosynthesis (2, 7, 15, 19, 23, 45). FbiA and FbiB are clearly involved in the conversion of FO into ... In Mycobacterium and Nocardia species, F420 is used by F420-dependent glucose-6-phosphate dehydrogenase (42, 43) and is required for activation of the experimental antituberculosis drug PA-824 by M. tuberculosis and Mycobacterium bovis strain BCG (hereafter referred to as M. bovis) (48). It is likely that other F420-dependent reactions will be discovered in mycobacteria, since genes corresponding to several proteins with homology to F420-dependent enzymes from other organisms are present in the M. tuberculosis genome (41). In Archaea, F420 is required for hydrogenase, formate, methylene-tetrahydromethanopterin, and alcohol dehydrogenases, methylene-tetrahydromethanopterin reductase, and quinone oxidoreductase (20, 22, 24, 28, 31, 50). F420 is used by Streptomyces for lincomycin and tetracycline biosynthesis (8, 29, 34, 46), and possibly in mitomycin C biosynthesis (32). The Archaea M. thermoautotrophicum and Halobacterium sp., the green alga Scenedesmus acutus, and the cyanobacterium Synechocystis sp. use F420 in photolyase (16, 17, 26, 38). In M. thermoautotrophicum, the pathway by which F420 is made has been studied with labeling (15, 23, 45) and enzymatic approaches (19), which have allowed development of a hypothetical pathway, an overview of which is shown in Fig. ​Fig.1B.1B. Due to our interest in F420 biosynthesis and in the metabolism of pathogenic mycobacteria, we have begun a study to identify the genes involved in F420 biosynthesis in Mycobacterium. The only description of genes that are required for F420 biosynthesis has been a recent report that homologues of the M. tuberculosis genes Rv3261 and Rv3262 are required for F420 biosynthesis in M. bovis (7). We named these genes fbiA and fbiB, respectively. Here we report that transposon Tn5367 insertion into the M. bovis homologue of the Rv1173 gene from M. tuberculosis creates mutants that cannot produce F420 or the biosynthetic intermediate FO. We name this gene fbiC (for F420 biosynthesis) and conclude that it is required for a step in the pathway prior to FO, before the deazaflavin ring is formed.

  • Large-scale production of Coenzyme F420-5,6 by using Mycobacterium smegmatis.
    Applied and environmental microbiology, 2002
    Co-Authors: Dale W. Isabelle, D. Randall Simpson, Lacy Daniels
    Abstract:

    Production of Coenzyme F420 and its biosynthetic precursor FO was examined with a variety of aerobic actinomycetes to identify an improved source for these materials. Based on fermentation costs, safety, and ease of growth, Mycobacterium smegmatis was the best source for F420-5,6. M. smegmatis produced 1 to 3 μmol of intracellular F420 per liter of culture, which was more than the 0.85 to 1.0 μmol of F420-2 per liter usually obtained with Methanobacterium thermoautotrophicum and ∼10-fold higher than what was previously reported for the best aerobic actinomycetes. An improved chromatography system using rapidly flowing quaternary aminoethyl ion-exchange material and Florisil was used to more quickly and easily purify F420 than with previous methods.

  • Structures of Coenzyme F420 in Mycobacterium species
    Archives of Microbiology, 2001
    Co-Authors: Thomas B. Bair, Dale W. Isabelle, Lacy Daniels
    Abstract:

    The structure of Coenzyme F420 in Mycobacterium smegmatis was examined using proton NMR, amino acid analysis, and HPLC. The two major F420 structures were shown to be composed of a chromophore identical to that of F420 from Methanobacterium thermoautotrophicum, with a side chain of a ribityl residue, a lactyl residue and five or six glutamate groups (F420–5 and F420–6). Peptidase treatment studies suggested that L-glutamate groups are linked by γ-glutamyl bonds in the side chain. HPLC analysis indicated that Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium fortuitum have F420–5 and F420–6 as the predominant structures, whereas Mycobacterium avium contains F420–5, F420–6 and F420–7 in significant amounts. 7,8-Didemethyl 8-hydroxy 5-deazariboflavin (FO), an intermediate in F420 biosynthesis, accounted for about 1–7% of the total deazaflavin in cells. Peptidase treatment of F420 created F420 derivatives that may be useful for the assay of enzymes involved in F420 biosynthesis.

  • si face stereospecificity at c5 of Coenzyme F420 for F420 dependent glucose 6 phosphate dehydrogenase from mycobacterium smegmatis and F420 dependent alcohol dehydrogenase from methanoculleus thermophilicus
    FEBS Journal, 1996
    Co-Authors: Andreas R Klein, Holger Berk, Endang Purwantini, Lacy Daniels, Rudolf K Thauer
    Abstract:

    Coenzyme F420 is a 5-deazaflavin. Upon reduction, 1,5-dihydro-Coenzyme F420 is formed with a prochiral center at C5. In this study we report that the F420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis and the F420-dependent alcohol dehydrogenase from Methanoculleus thermophilicus are Si -face stereospecific with respect to C5 of the 5-deazaflavin. These results were obtained by following the stereochemical course of the reversible incorporation of 3H into F420 from tritium-labeled substrates. Our findings bring to eight the number of Coenzyme-F420-dependent enzymes shown to be Si -face stereospecific. No F420-dependent enzyme with Re -face stereospecificity is known. This is noteworthy since Coenzyme F420 is functionally similar to pyridine nucleotides for which both Si -face and Re -face specific enzymes have been found.

  • Si‐Face Stereospecificity at C5 of Coenzyme F420 for F420‐Dependent Glucose‐6‐Phosphate Dehydrogenase from Mycobacterium smegmatis and F420‐Dependent Alcohol Dehydrogenase from Methanoculleus thermophilicus
    FEBS Journal, 1996
    Co-Authors: Andreas R Klein, Holger Berk, Endang Purwantini, Lacy Daniels, Rudolf K Thauer
    Abstract:

    Coenzyme F420 is a 5-deazaflavin. Upon reduction, 1,5-dihydro-Coenzyme F420 is formed with a prochiral center at C5. In this study we report that the F420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis and the F420-dependent alcohol dehydrogenase from Methanoculleus thermophilicus are Si -face stereospecific with respect to C5 of the 5-deazaflavin. These results were obtained by following the stereochemical course of the reversible incorporation of 3H into F420 from tritium-labeled substrates. Our findings bring to eight the number of Coenzyme-F420-dependent enzymes shown to be Si -face stereospecific. No F420-dependent enzyme with Re -face stereospecificity is known. This is noteworthy since Coenzyme F420 is functionally similar to pyridine nucleotides for which both Si -face and Re -face specific enzymes have been found.

Biswarup Mukhopadhyay - One of the best experts on this subject based on the ideXlab platform.

  • Coenzyme F420-Dependent Glucose-6-Phosphate Dehydrogenase-Coupled Polyglutamylation of Coenzyme F420 in Mycobacteria
    Journal of Bacteriology, 2018
    Co-Authors: Endang Purwantini, Usha Loganathan, Biswarup Mukhopadhyay
    Abstract:

    ABSTRACT Coenzyme F420 plays a key role in the redox metabolisms of various archaea and bacteria, including Mycobacterium tuberculosis. In M. tuberculosis, F420-dependent reactions have been linked to several virulence factors. F420 carries multiple glutamate residues in the side chain, forming F420-n species (n, number of glutamate residues), and the length of this side chain impacts cellular physiology. M. tuberculosis strains with F420 species carrying shorter side chains exhibit resistance to delamanid and pretomanid, two new tuberculosis (TB) drugs. Thus, the process of polyglutamylation of F420 is of great interest. It has been known from genetic analysis that in mycobacteria an F420-0 γ-glutamyl ligase (FbiB) introduces up to seven glutamate residues into F420. However, purified FbiB of M. tuberculosis (MtbFbiB) is either inefficient or incapable of incorporating more than two glutamates. We found that, in vitro, MtbFbiB synthesized side chains containing up to seven glutamate residues if F420 was presented to the enzyme in a two-electron reduced state (F420H2). Our genetic analysis in Mycobacterium bovis BCG and Mycobacterium smegmatis and an analysis of literature data on M. tuberculosis revealed that in these mycobacteria the polyglutamylation process requires the assistance of F420-dependent glucose-6-phosphate dehydrogenase (Fgd) which reduces F420 to F420H2. We hypothesize that, starting with F420-0H2, the amino-terminal domain of FbiB builds F420-2H2, which is then transferred to the carboxy-terminal domain for further glutamylation; F420-2H2 modifies the carboxy-terminal domain structurally to accommodate longer glutamyl chains. This system is analogous to folylpolyglutamate synthase, which introduces more than one glutamate residue into folate only after this vitamin is reduced to tetrahydrofolate. IMPORTANCE Coenzyme F420-dependent reactions of Mycobacterium tuberculosis, which causes tuberculosis, potentially contributes to the virulence of this bacterium. The Coenzyme carries a glutamic acid-derived tail, the length of which influences the metabolism of M. tuberculosis. Mutations that eliminate the production of F420 with longer tails make M. tuberculosis resistant to two new tuberculosis drugs. This report describes that the synthesis of longer glutamyl tails of F420 requires concerted actions of two enzymes, one of which reduces the Coenzyme prior to the action of the other, which catalyzes polyglutamylation. This knowledge will help to develop more effective tuberculosis (TB) drugs. Remarkably, the introduction of multiple glutamate residues into the sidechain of folate (vitamin B9) requires similar concerted actions, where one enzyme reduces the vitamin to tetrahydrofolate and the other catalyzes polyglutamylation; folate is required for DNA and amino acid synthesis. Thus, the reported research has also revealed a key similarity between two important cellular systems.

  • Coenzyme F420-Dependent Sulfite Reductase-Enabled Sulfite Detoxification and Use of Sulfite as a Sole Sulfur Source by Methanococcus maripaludis
    Applied and Environmental Microbiology, 2008
    Co-Authors: Eric F Johnson, Biswarup Mukhopadhyay
    Abstract:

    Coenzyme F420-dependent sulfite reductase (Fsr) of Methanocaldococcus jannaschii, a sulfite-tolerant methanogen, was expressed with activity in Methanococcus maripaludis, a sulfite-sensitive methanogen. The recombinant organism reduced sulfite to sulfide and grew with sulfite as the sole sulfur source, indicating that Fsr is a sulfite detoxification and assimilation enzyme for methanogens and that M. maripaludis synthesizes siroheme.

  • a new type of sulfite reductase a novel Coenzyme F420 dependent enzyme from the methanarchaeon methanocaldococcus jannaschii
    Journal of Biological Chemistry, 2005
    Co-Authors: Eric F Johnson, Biswarup Mukhopadhyay
    Abstract:

    Abstract Methanocaldococcus jannaschii is a hypertheromphilic, strictly hydrogenotrophic, methanogenic archaeon of ancient lineage isolated from a deep-sea hydrothermal vent. It requires sulfide for growth. Sulfite is inhibitory to the methanogens. Yet, we observed that M. jannaschii grows and produces methane with sulfite as the sole sulfur source. We found that in this organism sulfite induces a novel, highly active, Coenzyme F420-dependent sulfite reductase (Fsr) with a cell extract specific activity of 0.57 μmol sulfite reduced min-1 mg-1 protein. The cellular level of Fsr protein is comparable to that of methyl-Coenzyme M reductase, an enzyme essential for methanogenesis and a possible target for sulfite. Purified Fsr reduces sulfite to sulfide using reduced F420 (H2F420) as the electron source (Km: sulfite, 12 μm; H2F420, 21 μm). Therefore, Fsr provides M. jannaschii an anabolic ability and protection from sulfite toxicity. The N-terminal half of the 70-kDa Fsr polypeptide represents a H2F420 dehydrogenase and the C-terminal half a dissimilatory-type siroheme sulfite reductase, and Fsr catalyzes the corresponding partial reactions. Previously described sulfite reductases use nicotinamides and cytochromes as electron carriers. Therefore, this is the first report of a Coenzyme F420-dependent sulfite reductase. Fsr homologs were found only in Methanopyrus kandleri and Methanothermobacter thermautotrophicus, two strictly hydrogenotrophic thermophilic methanogens. fsr is the likely ancestor of H2F420 dehydrogenases, which serve as electron input units for membrane-based energy transduction systems of certain late evolving archaea, and dissimilatory sulfite reductases of bacteria and archaea. fsr could also have arisen from lateral gene transfer and gene fusion events.

  • cloning sequencing and transcriptional analysis of the Coenzyme F420 dependent methylene 5 6 7 8 tetrahydromethanopterin dehydrogenase gene from methanobacterium thermoautotrophicum strain marburg and functional expression in escherichia coli
    Journal of Biological Chemistry, 1995
    Co-Authors: Biswarup Mukhopadhyay, Endang Purwantini, T D Pihl, John N Reeve, Lacy Daniels
    Abstract:

    Abstract Two methylenetetrahydromethanopterin dehydrogenases have been purified from Methanobacterium thermoautotrophicum strain Marburg: one (MTD) is Coenzyme F420-dependent and oxygen-stable (Mukhopadhyay, B., and Daniels, L.(1989) Can. J. Microbiol. 35, 499-507), and the other (MTH) is Coenzyme F420-independent (or hydrogenase-type) and oxygen-sensitive (Zirngibl, C., Hedderich, R., and Thauer, R. K.(1990) FEBS Lett. 261, 112-116). Based on the NH 2-terminal sequence of MTD, a 36-mer oligonucleotide was designed and used to identify and clone a 6.1-kilobase pair EcoRI fragment of M. thermoautotrophicum DNA. Sequencing of this fragment revealed an 825-base pair (bp) MTD encoding gene (mtd), which was expressed in Escherichia coli yielding an enzyme that, like the native enzyme, was oxygen-stable, strictly dependent on Coenzyme F420, thermostable, thermophilic, and exhibited maximum activity at an acidic pH. The amino acid sequence predicts that MTD is a hydrophobic and acidic protein with no identifiable homology to MTH (von Bunau, R., Zirngibl, C., Thauer, R. K., and Klein, A.(1991) Eur. J. Biochem. 202, 1205-1208), but comparisons with Coenzyme F420 utilizing enzymes revealed a conserved region at the NH2 terminus of MTD that could correspond to the ability to interact with Coenzyme F420. The mtd transcript was ∼900 nucleotides long and initiated 8 bp upstream of the translation initiation codon and 22 bp downstream from an archaeal promoter sequence. The mtd coding sequence was followed by several poly(dT) sequences and an inverted repeat that could be transcription termination signals.

  • Characterization of F390 synthetase activity in cell extracts of Methanobacterium thermoautotrophicum Marburg
    Canadian Journal of Microbiology, 1994
    Co-Authors: Endang Purwantini, Biswarup Mukhopadhyay, Lacy Daniels
    Abstract:

    The F390 synthetase activity in cell extracts of Methanobacterium thermoautotrophicum Marburg increased by two times upon preincubation at 37 °C with its substrates, ATP (or GTP) and Coenzyme F420, but not with either of these compounds alone. In the 0–37 °C range, preincubation at 37 °C gave maximal enhancement in activity. F390 synthetase activity in cell extracts of strain Marburg was maximal at 45 °C, whereas F390 synthetase from M. thermoautotrophicum ΔH had maximal activity at 55 °C; both strains grew optimally at 65 °C. Data derived from the Arrhenius plot supported our earlier conclusion that the F390 synthetase activity of strain Marburg could lead to a loss of 70% of the available F420 during extraction of this factor from cells via an aerobic cell extract procedure even if the temperature was maintained at 4 °C.Key words: Methanobacterium thermoautotrophicum, F390 synthetase, activation, Coenzyme F420

Endang Purwantini - One of the best experts on this subject based on the ideXlab platform.

  • Coenzyme F420-Dependent Glucose-6-Phosphate Dehydrogenase-Coupled Polyglutamylation of Coenzyme F420 in Mycobacteria
    Journal of Bacteriology, 2018
    Co-Authors: Endang Purwantini, Usha Loganathan, Biswarup Mukhopadhyay
    Abstract:

    ABSTRACT Coenzyme F420 plays a key role in the redox metabolisms of various archaea and bacteria, including Mycobacterium tuberculosis. In M. tuberculosis, F420-dependent reactions have been linked to several virulence factors. F420 carries multiple glutamate residues in the side chain, forming F420-n species (n, number of glutamate residues), and the length of this side chain impacts cellular physiology. M. tuberculosis strains with F420 species carrying shorter side chains exhibit resistance to delamanid and pretomanid, two new tuberculosis (TB) drugs. Thus, the process of polyglutamylation of F420 is of great interest. It has been known from genetic analysis that in mycobacteria an F420-0 γ-glutamyl ligase (FbiB) introduces up to seven glutamate residues into F420. However, purified FbiB of M. tuberculosis (MtbFbiB) is either inefficient or incapable of incorporating more than two glutamates. We found that, in vitro, MtbFbiB synthesized side chains containing up to seven glutamate residues if F420 was presented to the enzyme in a two-electron reduced state (F420H2). Our genetic analysis in Mycobacterium bovis BCG and Mycobacterium smegmatis and an analysis of literature data on M. tuberculosis revealed that in these mycobacteria the polyglutamylation process requires the assistance of F420-dependent glucose-6-phosphate dehydrogenase (Fgd) which reduces F420 to F420H2. We hypothesize that, starting with F420-0H2, the amino-terminal domain of FbiB builds F420-2H2, which is then transferred to the carboxy-terminal domain for further glutamylation; F420-2H2 modifies the carboxy-terminal domain structurally to accommodate longer glutamyl chains. This system is analogous to folylpolyglutamate synthase, which introduces more than one glutamate residue into folate only after this vitamin is reduced to tetrahydrofolate. IMPORTANCE Coenzyme F420-dependent reactions of Mycobacterium tuberculosis, which causes tuberculosis, potentially contributes to the virulence of this bacterium. The Coenzyme carries a glutamic acid-derived tail, the length of which influences the metabolism of M. tuberculosis. Mutations that eliminate the production of F420 with longer tails make M. tuberculosis resistant to two new tuberculosis drugs. This report describes that the synthesis of longer glutamyl tails of F420 requires concerted actions of two enzymes, one of which reduces the Coenzyme prior to the action of the other, which catalyzes polyglutamylation. This knowledge will help to develop more effective tuberculosis (TB) drugs. Remarkably, the introduction of multiple glutamate residues into the sidechain of folate (vitamin B9) requires similar concerted actions, where one enzyme reduces the vitamin to tetrahydrofolate and the other catalyzes polyglutamylation; folate is required for DNA and amino acid synthesis. Thus, the reported research has also revealed a key similarity between two important cellular systems.

  • Lack of mycothiol and ergothioneine induces different protective mechanisms in Mycobacterium smegmatis.
    Biochemistry and biophysics reports, 2016
    Co-Authors: Arishma Rajkarnikar Singh, Endang Purwantini, Andrew Strankman, Ruzan Orkusyan, Mamta Rawat
    Abstract:

    Mycobacterium smegmatis contains the low molecular weight thiols, mycothiol (MSH) and ergothioneine (ESH). Examination of transposon mutants disrupted in mshC and egtA, involved in the biosynthesis of MSH and ESH respectively, demonstrated that both mutants were sensitive to oxidative, alkylating, and metal stress. However, the mshC mutant exhibited significantly more protein carbonylation and lipid peroxidation than wildtype, while the egtA mutant had less protein and lipid damage than wildtype. We further show that Ohr, KatN, and AhpC, involved in protection against oxidative stress, are upregulated in the egtA mutant. In the mshC mutant, an Usp and a putative thiol peroxidase are upregulated. In addition, mutants lacking MSH also contained higher levels of Coenzyme F420 as compared to wildtype and two Coenzyme F420 dependent enzymes were found to be upregulated. These results indicate that lack of MSH and ESH result in induction of different mechanisms for protecting against oxidative stress.

  • si face stereospecificity at c5 of Coenzyme F420 for F420 dependent glucose 6 phosphate dehydrogenase from mycobacterium smegmatis and F420 dependent alcohol dehydrogenase from methanoculleus thermophilicus
    FEBS Journal, 1996
    Co-Authors: Andreas R Klein, Holger Berk, Endang Purwantini, Lacy Daniels, Rudolf K Thauer
    Abstract:

    Coenzyme F420 is a 5-deazaflavin. Upon reduction, 1,5-dihydro-Coenzyme F420 is formed with a prochiral center at C5. In this study we report that the F420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis and the F420-dependent alcohol dehydrogenase from Methanoculleus thermophilicus are Si -face stereospecific with respect to C5 of the 5-deazaflavin. These results were obtained by following the stereochemical course of the reversible incorporation of 3H into F420 from tritium-labeled substrates. Our findings bring to eight the number of Coenzyme-F420-dependent enzymes shown to be Si -face stereospecific. No F420-dependent enzyme with Re -face stereospecificity is known. This is noteworthy since Coenzyme F420 is functionally similar to pyridine nucleotides for which both Si -face and Re -face specific enzymes have been found.

  • Si‐Face Stereospecificity at C5 of Coenzyme F420 for F420‐Dependent Glucose‐6‐Phosphate Dehydrogenase from Mycobacterium smegmatis and F420‐Dependent Alcohol Dehydrogenase from Methanoculleus thermophilicus
    FEBS Journal, 1996
    Co-Authors: Andreas R Klein, Holger Berk, Endang Purwantini, Lacy Daniels, Rudolf K Thauer
    Abstract:

    Coenzyme F420 is a 5-deazaflavin. Upon reduction, 1,5-dihydro-Coenzyme F420 is formed with a prochiral center at C5. In this study we report that the F420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis and the F420-dependent alcohol dehydrogenase from Methanoculleus thermophilicus are Si -face stereospecific with respect to C5 of the 5-deazaflavin. These results were obtained by following the stereochemical course of the reversible incorporation of 3H into F420 from tritium-labeled substrates. Our findings bring to eight the number of Coenzyme-F420-dependent enzymes shown to be Si -face stereospecific. No F420-dependent enzyme with Re -face stereospecificity is known. This is noteworthy since Coenzyme F420 is functionally similar to pyridine nucleotides for which both Si -face and Re -face specific enzymes have been found.

  • cloning sequencing and transcriptional analysis of the Coenzyme F420 dependent methylene 5 6 7 8 tetrahydromethanopterin dehydrogenase gene from methanobacterium thermoautotrophicum strain marburg and functional expression in escherichia coli
    Journal of Biological Chemistry, 1995
    Co-Authors: Biswarup Mukhopadhyay, Endang Purwantini, T D Pihl, John N Reeve, Lacy Daniels
    Abstract:

    Abstract Two methylenetetrahydromethanopterin dehydrogenases have been purified from Methanobacterium thermoautotrophicum strain Marburg: one (MTD) is Coenzyme F420-dependent and oxygen-stable (Mukhopadhyay, B., and Daniels, L.(1989) Can. J. Microbiol. 35, 499-507), and the other (MTH) is Coenzyme F420-independent (or hydrogenase-type) and oxygen-sensitive (Zirngibl, C., Hedderich, R., and Thauer, R. K.(1990) FEBS Lett. 261, 112-116). Based on the NH 2-terminal sequence of MTD, a 36-mer oligonucleotide was designed and used to identify and clone a 6.1-kilobase pair EcoRI fragment of M. thermoautotrophicum DNA. Sequencing of this fragment revealed an 825-base pair (bp) MTD encoding gene (mtd), which was expressed in Escherichia coli yielding an enzyme that, like the native enzyme, was oxygen-stable, strictly dependent on Coenzyme F420, thermostable, thermophilic, and exhibited maximum activity at an acidic pH. The amino acid sequence predicts that MTD is a hydrophobic and acidic protein with no identifiable homology to MTH (von Bunau, R., Zirngibl, C., Thauer, R. K., and Klein, A.(1991) Eur. J. Biochem. 202, 1205-1208), but comparisons with Coenzyme F420 utilizing enzymes revealed a conserved region at the NH2 terminus of MTD that could correspond to the ability to interact with Coenzyme F420. The mtd transcript was ∼900 nucleotides long and initiated 8 bp upstream of the translation initiation codon and 22 bp downstream from an archaeal promoter sequence. The mtd coding sequence was followed by several poly(dT) sequences and an inverted repeat that could be transcription termination signals.