Biosynthesis of coenzyme F420 and methanopterin in Methanobacterium thermoautotrophicum

Growing cultures of Methanobacterium thermoautotrophicum were supplemented with [U-¹⁴C]adenosine or [1-¹⁴C]adenosine. 7,8-Didemethyl-8-hydroxy-5-deazariboflavin (factor F₀) and 7-methylpterin were isolated from the culture medium. Hydrolysis of cellular RNA yielded purine and pyrimidine nucleotides. The ribose side chain of proffered adenosine is efficiently incorporated into cellular adenosine and guanosine nucleotide pools but not into pyrimidine nucleotides. Thus, M. thermoautotrophicum can utilize exogenous adenosine by direct phosphorylation without hydrolysis of the glycosidic bond, and AMP can be efficiently converted to GMP. Factor F₀ and 7-methylpterin had approximately the same specific activities as the purine nucleotides. It follows that the ribityl side chain of factor F₀ is derived from the ribose side chain of a nucleotide precursor by reduction. The pyrazine ring of methanopterin is formed by ring expansion involving the ribose side chain of the precursor, GTP.Abbreviations Factor F₀ 8-hydroxy-6,7-didemethyl-5-deazariboflavin - APRT adenine phosphoribosyltransferase - GPRT guanine phosphoribosyltransferase - PRPP phosphoribosylpyrophosphate - HPLC high performance liquid chromatography     

Structures of coenzyme F(420) in Mycobacterium species

The structure of coenzyme F(420) in Mycobacterium smegmatis was examined using proton NMR, amino acid analysis, and HPLC. The two major F(420) structures were shown to be composed of a chromophore identical to that of F(420) from Methanobacterium thermoautotrophicum, with a side chain of a ribityl residue, a lactyl residue and five or six glutamate groups (F(420)-5 and F(420)-6). Peptidase treatment studies suggested that L-glutamate groups are linked by gamma-glutamyl bonds in the side chain. HPLC analysis indicated that Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium fortuitum have F(420)-5 and F(420)-6 as the predominant structures, whereas Mycobacterium avium contains F(420)-5, F(420)-6 and F(420)-7 in significant amounts. 7,8-Didemethyl 8-hydroxy 5-deazariboflavin (FO), an intermediate in F(420) biosynthesis, accounted for about 1-7% of the total deazaflavin in cells. Peptidase treatment of F(420) created F(420) derivatives that may be useful for the assay of enzymes involved in F(420) biosynthesis.     

Immunogold localization of coenzyme F420-reducing formate dehydrogenase and coenzyme F420-reducing hydrogenase in Methanobacterium formicicum

The ultrastructural locations of the coenzyme F₄₂₀-reducing formate dehydrogenase and coenzyme F₄₂₀-reducing hydrogenase of Methanobacterium formicicum were determined using immunogold labeling of thin-sectioned, Lowicryl-embedded cells. Both enzymes were located predominantly at the cell membrane. Whole cells displayed minimal F₄₂₀-dependent formate dehydrogenase activity or F₄₂₀-dependent hydrogenase activity, and little activity was released upon osmotic shock treatment, suggesting that these enzymes are not soluble periplasmic proteins. Analysis of the deduced amino acid sequences of the formate dehydrogenase subunits revealed no hydrophobic regions that could qualify as putative membrane-spanning domains.Abbreviation PBST Phosphate-buffered saline containing 0.1% (v/v) Triton X-100     

Structure of coenzyme F(420) dependent methylenetetrahydromethanopterin reductase from two methanogenic archaea

Coenzyme F(420)-dependent methylenetetrahydromethanopterin reductase (Mer) is an enzyme of the Cl metabolism in methanogenic and sulfate reducing archaea. It is composed of identical 35-40 kDa subunits and lacks a prosthetic group. The crystal structure of Mer from Methanopyrus kandleri (kMer) revealed in one crystal form a dimeric and in another a tetrameric oligomerisation state and that from Methanobacterium thermoautotrophicum (tMer) a dimeric state. Each monomer is primarily composed of a TIM-barrel fold enlarged by three insertion regions. Insertion regions 1 and 2 contribute to intersubunit interactions. Insertion regions 2 and 3 together with the C-terminal end of the TIM-barrel core form a cleft where the binding sites of coenzyme F(420) and methylene-tetrahydromethanopterin are postulated. Close to the coenzyme F(420)-binding site lies a rarely observed non-prolyl cis-peptide bond. It is surprising that Mer is structurally most similar to a bacterial FMN-dependent luciferase which contains a non-prolyl cis-peptide bond at the equivalent position. The structure of Mer is also related to that of NADP-dependent FAD-harbouring methylenetetrahydrofolate reductase (MetF). However, Mer and MetF do not show sequence similarities although they bind related substrates and catalyze an analogous reaction.     

F420H2 oxidase (FprA) from Methanobrevibacter arboriphilus, a coenzyme F420-dependent enzyme involved in O2 detoxification

Cell suspensions of Methanobrevibacter arboriphilus catalyzed the reduction of O₂ with H₂ at a maximal specific rate of 0.4 U (mol/min) per mg protein with an apparent K _m for O₂ of 30 M. 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 K _m for F₄₂₀ was 30 M and that for O₂ was 2 M with a V _max 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.Dedicated to Hans Schlegel on the occasion of his 80th birthday.     

Molecular insights into the biosynthesis of the F420 coenzyme

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.     

Studies on the biosynthesis of coenzyme F420 in methanogenic bacteria

Coenzyme F₄₂₀ is a 8-hydroxy-5-deazaflavin present in methanogenic bacteria. We have investigated whether the pyrimidine ring of the deazaflavin originates from guanine as in flavin biosynthesis, in which the pyrimidine ring of guanine is conserved. For this purpose the incorporation of [2-¹⁴C]guanine and of [8-¹⁴C]guanine into F₄₂₀ by growing cultures of Methanobacterium thermoautotrophicum was studied. Only in the case of [2-¹⁴C]guanine did F₄₂₀ become labeled. The specific radioactivity of the deazaflavin and of guanine isolated from nucleic acids of [2-¹⁴C]guanine grown cells were identical. This finding suggests that the pyrimidine ring of the deazaflavin and of flavins are synthesized by the same pathway.F₄₂₀ did not become labeled when M. thermoautotrophicum was grown in the presence of methyl-[¹⁴C] methionine, [U-¹⁴C]phenylalanine or [U-¹⁴C]tyrosine. This excludes that C-5 of the deazaflavin is derived from the methyl group of methionine and that the benzene ring comes from phenylalanine or tyrosine.     

Characterization of the 2-phospho-L-lactate transferase enzyme involved in coenzyme F(420) biosynthesis in Methanococcus jannaschii

The protein product of the Methanococcus jannaschii MJ1256 gene has been expressed in Escherichia coli, purified to homogeneity, and shown to be involved in coenzyme F(420) biosynthesis. The protein catalyzes the transfer of the 2-phospholactate moiety from lactyl (2) diphospho-(5')guanosine (LPPG) to 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo) with the formation of the L-lactyl phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F(420)-0) and GMP. On the basis of the reaction catalyzed, the enzyme is named LPPG:Fo 2-phospho-L-lactate transferase. Since the reaction is the fourth step in the biosynthesis of coenzyme F(420), the enzyme has been designated as CofD, the product of the cofD gene. The transferase requires Mg(2+) for activity, and the catalysis does not appear to proceed via a covalent intermediate. To a lesser extent CofD also catalyzes a number of additional reactions that include the formation of Fo-P, when the enzyme is incubated with Fo and GDP, GTP, pyrophosphate, or tripolyphosphate, and the hydrolysis of F(420)-0 to Fo. All of these side reactions can be rationalized as occurring by a common mechanism. CofD has no recognized sequence similarity to any previously characterized enzyme.     

CofE catalyzes the addition of two glutamates to F420-0 in F420 coenzyme biosynthesis in Methanococcus jannaschii

The protein product of the Methanococcus jannaschii MJ0768 gene has been expressed in Escherichia coli, purified to homogeneity, and shown to catalyze the GTP-dependent addition of two l-glutamates to the l-lactyl phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin (F(420)-0) to form F(420)-0-glutamyl-glutamate (F(420)-2). Since the reaction is the fifth step in the biosynthesis of coenzyme F(420), the enzyme has been designated as CofE, the product of the cofE gene. Gel filtration chromatography indicates CofE is a dimer. The enzyme has no recognized sequence similarity to any previously characterized proteins. The enzyme has an absolute requirement for a divalent metal ion and a monovalent cation. Among the metal ions tested, a mixture of Mn(2+), Mg(2+), and K(+) is the most effective. CofE catalyzes amide bond formation with the cleavage of GTP to GDP and inorganic phosphate, likely involving the activation of the free carboxylate group of F(420)-0 to give an acyl phosphate intermediate. Evidence for the occurrence of this intermediate is presented. A reaction mechanism for the enzyme is proposed and compared with other members of the ADP-forming amide bond ligase family.     

Relation of coenzyme F420 to the activity of methanogenic microorganisms

Summary The relationship between the coenzyme F₄₂₀ content and the activity of methanogenic microorganisms was investigated under different cultivation conditions in anaerobic reactors. The coenzyme F₄₂₀ concentration depends on the substrate used and the cultivation conditions. Coenzyme F₄₂₀ appears not to be a measure of the total methanogenic activity but rather a measure of the amount of methanogenic microorganisms in mixed anaerobic cultures.     

Roles of coenzyme F420-reducing hydrogenases and hydrogen- and F420-dependent methylenetetrahydromethanopterin dehydrogenases in reduction of F420 and production of hydrogen during methanogenesis

Reduced coenzyme F420 (F420H2) is an essential intermediate in methanogenesis from CO2. During methanogenesis from H2 and CO2, F420H2 is provided by the action of F420-reducing hydrogenases. However, an alternative pathway has been proposed, where H2-dependent methylenetetrahydromethanopterin dehydrogenase (Hmd) and F420H2-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd) together reduce F420 with H2. Here we report the construction of mutants of Methanococcus maripaludis that are defective in each putative pathway. Their analysis demonstrates that either pathway supports growth on H2 and CO2. Furthermore, we show that during growth on formate instead of H2, where F420H2 is a direct product of formate oxidation, H2 production occurs. H2 presumably arises from the oxidation of F420H2, and the analysis of the mutants during growth on formate suggests that this too can occur by either pathway. We designate the alternative pathway for the interconversion of H2 and F420H2 the Hmd-Mtd cycle.     

Improved assay of coenzyme F420 analogues from methanogenic bacteria

Summary An improved method for separating analogues of coenzyme F₄₂₀ by isocratic reversed-phase high performance liquid chromatography is described. The method offers improved resolution, shorter chromatography runs and requires less complex apparatus.     

Ammonia assimilation and glutamate incorporation in coenzyme F420 derivatives ofMethanosarcina barkeri

Methanosarcina barkeri was able to grow on L-alanine and L-glutamate as sole nitrogen sources. Cell yields were 0.5 g/l and 0.7 g/l (wet wt), respectively. The mechanism of ammonia assimilation inMethanosarcina barkeri strain MS was studied by analysis of enzyme activities. Activity levels of nitrogen-assimilating enzymes in extracts of cells grown on different nitrogen sources (ammonia, 0.05–100 mM; L-alanine, 10 mM; L-glutamate, 10 mM) were compared. Activities of glutamate dehydrogenase, glutamate synthase, glutamine synthetase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase could be measured in cells grown on these three nitrogen sources. Alanine dehydrogenase was not detected under the growth conditions used. None of the measured enzyme activities varied significantly in response to the NH₄ ⁺ concentration. The length of the poly--glutamyl side chain of F₄₂₀ derivatives turned out to be independent of the concentration of ammonia in the culture medium.Abbreviations ADH alanine dehydrogenase - FO 7,8-didemethyl-8-hydroxy-5-deazariboflavin - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase - GS glutamine synthetase - H₄MPT tetrahydromethanopterin     

Identification and characterization of the 2-phospho-L-lactate guanylyltransferase involved in coenzyme F420 biosynthesis

Coenzyme F 420 is a hydride carrier cofactor functioning in methanogenesis. One step in the biosynthesis of coenzyme F 420 involves the coupling of 2-phospho- l-lactate (LP) to 7,8-didemethyl-8-hydroxy-5-deazaflavin, the F 420 chromophore. This condensation requires an initial activation of 2-phospho- l-lactate through a pyrophosphate linkage to GMP. Bioinformatic analysis identified an uncharacterized archaeal protein in the Methanocaldococcus jannaschii genome, MJ0887, which could be involved in this transformation. The predicted MJ0887-derived protein has domain similarity with other known nucleotidyl transferases. The MJ0887 gene was cloned and overexpressed, and the purified protein was found to catalyze the formation of lactyl-2-diphospho-5'-guanosine from LP and GTP. Kinetic constants were determined for the MJ0887-derived protein with both LP and GTP substrates and are as follows: V max = 3 micromol min (-1) mg (-1), GTP K M (app) = 56 microM, and k cat/ K M (app) = 2 x 10 (4) M (-1) s (-1) and LP K M (app) = 36 microM, and k cat/ K M (app) = 4 x 10 (4) M (-1) s (-1). The MJ0887 gene product has been designated CofC to indicate its involvement in the third step of coenzyme F 420 biosynthesis.     

Immunocytochemical localization of the coenzyme F420-reducing hydrogenase in Methanosarcina barkeri Fusaro.

The cytological localization of the 8-hydroxy-5-deazaflavin (coenzyme F420)-reducing hydrogenase of Methanosarcina barkeri Fusaro was determined by immunoelectron microscopy, using a specific polyclonal rabbit antiserum raised against the homogeneous deazaflavin-dependent enzyme. In Western blot (immunoblot) experiments this antiserum reacted specifically with the native coenzyme F420-reducing hydrogenase, but did not cross-react with the coenzyme F420-nonreducing hydrogenase activity also detectable in crude extracts prepared from methanol-grown Methanosarcina cells. Immunogold labelling of ultrathin sections of anaerobically fixed methanol-grown cells from the exponential growth phase revealed that the coenzyme F420-reducing hydrogenase was predominantly located in the vicinity of the cytoplasmic membrane. From this result we concluded that the deazaflavin-dependent hydrogenase is associated with the cytoplasmic membrane in intact cells of M. barkeri during growth on methanol as the sole methanogenic substrate, and a possible role of this enzyme in the generation of the electrochemical proton gradient is discussed.     

Mechanistic studies of the coenzyme F420 reducing formate dehydrogenase from Methanobacterium formicicum

Mechanistic studies have been undertaken on the coenzyme F420 dependent formate dehydrogenase from Methanobacterium formicicum. The enzyme was specific for the si face hydride transfer to C5 of F420 and joins three other F420-recognizing methanogen enzymes in this stereospecificity, consistent perhaps with a common type of binding site for this 8-hydroxy-5-deazariboflavin. While catalysis probably occurs by hydride transfer from formate to the enzyme to generate an EH2 species and then by hydride transfer back out to F420, the formate-derived hydrogen exchanged with solvent protons before transfer back out to F420. The kinetics of hydride transfer from formate revealed that this step is not rate determining, which suggests that the rate-determining step is an internal electron transfer. The deflavo formate dehydrogenase was amenable to reconstitution with flavin analogues. The enzyme was sensitive to alterations in FAD structure in the 6-, 7-, and 8-loci of the benzenoid moiety in the isoalloxazine ring.     

Methanococcus jannaschii coenzyme F420 analogs contain a terminal alpha-linked glutamate

Analyses of the F(420)s present in Methanococcus jannaschii have shown that these cells contain a series of gamma-glutamyl-linked F(420)s capped with a single, terminal alpha-linked L-glutamate. The predominant form of F(420) was designated as alpha-F(420)-3 and represented 86% of the F(420)s in these cells. Analyses of Methanosarcina thermophila, Methanosarcina barkeri, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidus, and Mycobacterium smegmatis showed that they contained only gamma-glutamyl-linked F(420)s.     

Composition of the coenzyme F420-dependent formate dehydrogenase from Methanobacterium formicicum.

The coenzyme F420-dependent formate dehydrogenase from Methanobacterium formicicum was purified to electrophoretic homogeneity by anoxic procedures which included the addition of azide, flavin adenine dinucleotide (FAD), glycerol, and 2-mercaptoethanol to all buffer solutions to stabilize activity. The enzyme contains, in approximate molar ratios, 1 FAD molecule and 1 molybdenum, 2 zinc, 21 to 24 iron, and 25 to 29 inorganic sulfur atoms. Denaturation of the enzyme released a molybdopterin cofactor. The enzyme has a molecular weight of 177,000 and consists of one each of two different subunits, giving the composition alpha 1 beta 1. The molecular weight of the alpha-subunit is 85,000, and that of the beta-subunit is 53,000. The UV-visible spectrum is typical of nonheme iron-sulfur flavoprotein. Reduction of the enzyme facilitated dissociation of FAD, and the FAD-depleted enzyme was unable to reduce coenzyme F420. Preincubation of the FAD-depleted enzyme with FAD restored coenzyme F420-dependent activity.     

Demonstration that fbiC is required by Mycobacterium bovis BCG for coenzyme F(420) and FO biosynthesis

Using the nitroimidazopyran-based antituberculosis drug PA-824 as a selective agent, transposon-generated Mycobacterium bovis strain BCG (M. bovis) mutants that could not make coenzyme F(420) were identified. Four independent mutants that could not make F(420) or the biosynthesis intermediate FO were examined more closely. These mutants contained transposons inserted in the M. bovis homologue of the Mycobacterium tuberculosis gene Rv1173, which we have named fbiC. Complementation of an M. bovis FbiC(-) mutant with fbiC restored the F(420) phenotype. These data demonstrate that fbiC is essential for F(420) production and that FbiC participates in a portion of the F(420) biosynthetic pathway between pyrimidinedione and FO. Homologues of fbiC were found in all 11 microorganisms that have been fully sequenced and that are known to make F(420). Four of these homologues (all from members of the aerobic actinomycetes) coded for proteins homologous over the entire length of the M. bovis FbiC, but in seven microorganisms two separate genes were found to code for proteins homologous with either the N-terminal or C-terminal portions of the M. bovis FbiC. Histidine-tagged FbiC overexpressed in Escherichia coli produced a fusion protein of the molecular mass predicted from the M. bovis BCG sequence (approximately 95,000 Da), as well as three other histidine-tagged proteins of significantly smaller size, which are thought to be proteolysis products of the FbiC fusion protein.     

Distribution of coenzyme F420 and properties of its hydrolytic fragments.

The ability of hydrolytic products of coenzyme F420 to substitute for F420 in the hydrogenase and nicotinamide adenine dinucleotide phosphate-liniked hydrogenase systems of Methanobacterium strain M.o.H. was kinetically determined. The nicotinamide adenine dinucleotide phosphate-linked hydrogenase system was employed to quantitate the levels of F420 in a number of methanogenic bacteria as well as in some nonmethanogens. Methanobacterium ruminantium and Methanosarcina barkeri contained low levels of F420, whereas other methanogens tested contained high levels (100 to 400 mg/kg of cells). F420 from six of the seven methanogens was tested by thin-layer electrophoresis and was found to be electrophoretically identical to that purified from Methanobacterium strain M.o.H. The only exception was M. barkeri, which contained a more electronegative derivative of F420. Acetobacterium woodii, Escherichia coli, and yeast extract contained no compounds able to substitute for F420 in the nicotinamide adenine dinucleotide phosphate-linked hydrogenase system.