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.     

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

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.     

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.     

Biosynthesis of the phosphodiester bond in coenzyme F(420) in the methanoarchaea

The biochemical route for the formation of the phosphodiester bond in coenzyme F(420), one of the methanogenic coenzymes, has been established in the methanoarchaea Methanosarcina thermophila and Methanococcus jannaschii. The first step in the formation of this portion of the F(420) structure is the GTP-dependent phosphorylation of L-lactate to 2-phospho-L-lactate and GDP. The 2-phospho-L-lactate represents a new natural product that was chemically identified in Methanobacterium thermoautotrophicum, M. thermophila, and Mc. jannaschii. Incubation of cell extracts of both M. thermophila and Mc. jannaschii with [hydroxy-(18)O, carboxyl-(18)O(2)]lactate and GTP produced 2-phospho-L-lactate with the same (18)O distribution as found in both the starting lactate and the lactate recovered from the incubation. These results indicate that the carboxyl oxygens are not involved in the phosphorylation reaction. Incubation of Sephadex G-25 purified cell extracts of M. thermophila or Mc. jannaschii with 7,8-didemethyl-8-hydroxy-5-deazariboflavin (Fo), 2-phospho-L-lactate, and GTP or ATP lead to the formation of F(420)-0 (F(420) with no glutamic acids). This transformation was shown to involve two steps: (i) the GTP- or ATP-dependent activation of 2-phospho-L-lactate to either lactyl(2)diphospho-(5')guanosine (LPPG) or lactyl(2)diphospho-(5')adenosine (LPPA) and (ii) the reaction of the resulting LPPG or LPPA with Fo to form F(420)-0 with release of GMP or AMP. Attempts to identify LPPG or LPPA intermediates by incubation of cell extracts with L-[U-(14)C]lactate, [U-(14)C]2-phospho-L-lactate, or [8-(3)H]GTP were not successful owing to the instability of these compounds toward hydrolysis. Synthetically prepared LPPG and LPPA had half-lives of 10 min at 50 degrees C (at pH 7.0) and decomposed into GMP or AMP and 2-phospho-L-lactate via cyclic 2-phospho-L-lactate. No evidence for the functioning of the cyclic 2-phospho-L-lactate in the in vitro biosynthesis could be demonstrated. Incubation of cell extracts of M. thermophila or Mc. jannaschii with either LPPG or LPPA and Fo generated F(420)-0. In summary, this study demonstrates that the formation of the phosphodiester bond in coenzyme F(420) follows a reaction scheme like that found in one of the steps of the DNA ligase reaction and in the biosynthesis of coenzyme B(12) and phospholipids.     

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.     

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.     

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.     

Purification and characterization of F420H2-dehydrogenase from Methanolobus tindarius.

The oxidation of F420H2 (reduced coenzyme F420) is a key reaction in the final step of methanogenesis. This step is catalyzed in Methanolobus tindarius by the membrane-bound F420H2-dehydrogenase which was purified 31-fold to apparent homogeneity. The apparent molecular mass of the native enzyme was 120 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of five different subunits of apparent molecular masses of 45 kDa, 40 kDa, 22 kDa, 18 kDa and 17 kDa. The purified F420H2-dehydrogenase, which was yellowish, contained 16 +/- 2 mol iron and 16 +/- 3 mol acid-labile sulfur/mol enzyme. No flavin could be detected. The oxygen-stable enzyme catalyzed the oxidation of F420H2 (apparent Km = 5.4 microM) with methylviologen and metronidazole as electron acceptors at a specific rate of 13 mumol.min-1.mg-1 (kcat = 25.5 s-1). The isoelectric point was at pH 5.0. The temperature optimum was at 37 degrees C and the pH optimum at 6.8.     

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.     

Occurrence of coenzyme F420 and its gamma-monoglutamyl derivative in nonmethanogenic archaebacteria.

Analysis of the fluorescent compounds extracted from six different species of halobacteria and one species each of Sulfolobus and Thermoplasma revealed the universal occurrence of coenzyme F420, (N-[N-[O-[5-(8-hydroxy-5-deazaisoalloxazin-10-yl)-2,3,4-trihydroxy -4-pentoxyhydroxyphosphinyl]-L-lactyl]-L-gamma-glutamyl]-L -glutamic acid), or its gamma-monoglutamyl derivative or both. The total amount (approximately 100 pmol/mg [dry weight]) of these compounds found in the halobacteria studied was approximately 5% of the amount previously reported for methanogenic bacteria. The amount of F420 found in the Sulfolobus and Thermoplasma strains was approximately 1% of that found in the halobacteria. The major compound in all but one of the examined strains was the gamma-monoglutamyl derivative of F420; one strain of halobacteria contained only F420. For the halobacterium-derived samples, the additional glutamic acid was shown to be linked by a gamma-glutamyl peptide bond to the terminal glutamic acid of the F420 core structure by enzymatic hydrolysis of the samples with three different gamma-glutamyltranspeptidases. The product of this enzymatic hydrolysis was F420 with one less glutamic acid in the side chain.     

Si-face stereospecificity at C5 of coenzyme F420 for F420H2 oxidase from methanogenic Archaea as determined by mass spectrometry

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.     

Structural aspects and immunolocalization of the F420-reducing and non-F420-reducing hydrogenases from Methanobacterium thermoautotrophicum Marburg.

The F420-reducing hydrogenase and the non-F420-reducing hydrogenase (EC 1.12.99.1.) were isolated from a crude extract of Methanobacterium thermoautotrophicum Marburg. Electron microscopy of the negatively stained F420-reducing hydrogenase revealed that the enzyme is a complex with a diameter of 15.6 nm. It consists of two ring-like, stacked, parallel layers each composed of three major protein masses arranged in rotational symmetry. Each of these masses appeared to be subdivided into smaller protein masses. Electron microscopy of negatively stained samples taken from intermediate steps of the purification process revealed the presence of enzyme particles bound to inside-out membrane vesicles. Linker particles of 10 to 20 kDa which mediate the attachment of the hydrogenase to the cytoplasmic membrane were seen. Immunogold labelling confirmed that the F420-reducing hydrogenase is a membrane-bound enzyme. Electron microscopy of the negatively stained purified non-F420-reducing hydrogenase revealed that the enzyme is composed of three subunits exhibiting different diameters (5, 4, and 2 to 3 nm). According to immunogold labelling experiments, approximately 70% of the non-F420-reducing hydrogenase protein molecules were located at the cell periphery; the remaining 30% were cytoplasmic. No linker particles were observed for this enzyme.     

The F420H2 dehydrogenase from Methanosarcina mazei is a Redox-driven proton pump closely related to NADH dehydrogenases

The F(420)H(2) dehydrogenase is part of the energy conserving electron transport system of the methanogenic archaeon Methanosarcina mazei G?1. Here it is shown that cofactor F(420)H(2)-dependent reduction of 2-hydroxyphenazine as catalyzed by the membrane-bound enzyme is coupled to proton translocation across the cytoplasmic membrane, exhibiting a stoichiometry of 0.9 H(+) translocated per two electrons transferred. The electrochemical proton gradient thereby generated was shown to drive ATP synthesis from ADP + P(i). The gene cluster encoding the F(420)H(2) dehydrogenase of M. mazei G?1 comprises 12 genes that are referred to as fpoA, B, C, D, H, I, J, K, L, M, N, and O. Analysis of the deduced amino acid sequences revealed that the enzyme is closely related to proton translocating NADH dehydrogenases of respiratory chains from bacteria (NDH-1) and eukarya (complex I). Like the NADH-dependent enzymes, the F(420)H(2) dehydrogenase is composed of three subcomplexes. The gene products FpoA, H, J, K, L, M, and N are highly hydrophobic and are homologous to subunits that form the membrane integral module of NDH-1. FpoB, C, D, and I have their counterparts in the amphipathic membrane-associated module of NDH-1. Homologues to the hydrophilic NADH-oxidizing input module are not present in M. mazei G?1. Instead, the gene product FpoF may be responsible for F(420)H(2) oxidation and may function as the electron input part. Thus, the F(420)H(2) dehydrogenase from M. mazei G?1 resembles eukaryotic and bacterial proton translocating NADH dehydrogenases in many ways. The enzyme from the methanogenic archaeon functions as a NDH-1/complex I homologue and is equipped with an alternative electron input unit for the oxidation of reduced cofactor F(420) and a modified output module adopted to the reduction of methanophenazine.     

Factor 420-dependent tyridine nucleotide-linked hydrogenase system of Methanobacterium ruminantium.

Methanobacterium ruminantium was shown to possess a nicotinamide adenine dinucleotide phosphate (NADP)-linked factor 420 (F420)-dependent hydrogenase system. This system was also shown to be present in Methanobacterium strain MOH. The hydrogenase system of M. ruminantium also links directly to F420, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), methyl viologen, and Fe-3 plus. It has a pH optimum of about 8 and an apparent Km for F420 of about 5 x 10-6 M at pH 8 when NADP is the electron acceptor. The F420-NADP oxidoreductase activity is inactive toward nicotinamide adenine dinucleotide (nad) and no NADPH:NAD or FADH2(FMNH2):NAD transhydrogenase system was detected. Neither crude ferredoxin nor boiled crude extract of Clostridium pasteuranum could replace F420 in the NADP-linked hydrogenase reaction of M. ruminantium. Also, neitther F420 nor a curde "ferredoxin" fraction from M. ruminantium extracts could substitute for ferredoxin in the pyruvate-ferredoxin oxidoreductase reaction of C. pasteurianum.     

Coenzyme F420 dependence of the methylenetetrahydromethanopterin dehydrogenase of Methanobacterium thermoautotrophicum

To identify the electron acceptor of the methylenetetrahydromethanopterin dehydrogenase of Methanobacterium thermoautotrophicum, we have purified the enzyme to homogeneity. The purified enzyme is absolutely dependent on coenzyme F420 (a 7,8-didemethyl-8-hydroxy-5-deazariboflavin derivative) for activity. Several alternative electron acceptors are ineffectual in the reaction. Changes in the absorption spectra of reaction mixtures indicate that 1.1 mol of coenzyme F420 is reduced per mol of substrate oxidized. The reaction is reversible and the equilibrium favors oxidation of methylenetetrahydromethanopterin.     

Membrane-bound F420H2-dependent heterodisulfide reduction in Methanococcus voltae

Washed membranes prepared from H₂+CO₂- or formate-grown cells of Methanococcus voltae catalyzed the oxidation of coenzyme F₄₂₀H₂ and the reduction of the heterodisulfide (CoB–S–S–CoM) of 2-mercaptoethanesulfonate and 7-mercaptoheptanoylthreonine phosphate, which is the terminal electron acceptor of the methanogenic pathway. The reaction followed a 1:1 stoichiometry according to the equation: F₄₂₀H₂ + COB–S–S–CoM → F₄₂₀ + CoM–SH + CoB–SH. These findings indicate that the reaction depends on a membrane-bound F₄₂₀H₂-oxidizing enzyme and on the heterodisulfide reductase, which remains partly membrane-bound after cell lysis. To elucidate the nature of the F₄₂₀H₂-oxidizing protein, washed membranes were solubilized with detergent, and the enzyme was purified by sucrose density centrifugation, anion-exchange chromatography, and gel filtration. Several lines of evidence indicate that F₄₂₀H₂ oxidation is catalyzed by a membrane-associated F₄₂₀-reducing hydrogenase. The purified protein catalyzed the H₂-dependent reduction of methyl viologen and F₄₂₀. The apparent molecular mass and the subunit composition (43, 37, and 27 kDa) are almost identical to those of the F₄₂₀-reducing hydrogenase that has already been purified from Mc. voltae. Moreover, the N-terminus of the 37-kDa subunit is identical to the amino acid sequence deduced from the fruG gene of the operon encoding the selenium-containing F₄₂₀-reducing hydrogenase from Mc. voltae. A distinct F₄₂₀H₂ dehydrogenase, which is present in methylotrophic methanogens, was not found in this organism. Received: 18 September 1998 / Accepted: 2 November 1998     

Structure of the F420H2:quinone oxidoreductase of Archaeoglobus fulgidus identification and overproduction of the F420H2-oxidizing subunit.

The F420H2:quinone oxidoreductase from the sulfate-reducing archaeon Archaeoglobus fulgidus is encoded by the fqo gene cluster which comprises 11 genes (fqo J, K, M, L, N, A, BC, D, H, I, F). The last gene of the cluster, fqoF, was overexpressed in Escherichia coli. The purified subunit was able to oxidize reduced cofactor F420 using the electron-acceptor system methyl viologen plus metronidazole. The specific activity at 78 degrees C was 64 micromol F420H2 oxidized. min-1.(mg protein)-1. The purified polypeptide contained 10.6 mol non-heme iron, 7.2 mol acid-labile sulfur and 0.7 mol FAD per mol protein. With the exception of fqoF, the deduced amino-acid sequences of all other genes show homologies to distinct subunits of NADH-quinone oxidoreductases from prokaryotes and eukaryotes. Thus, it is concluded that the F420H2-dependent and the NADH-dependent enzyme are functional equivalents. Both proteins are the initial enzymes of membrane-bound electron-transport systems and are involved in energy conservation. In parallel with bacterial complex I, the F420H2:quinone oxidoreductase may be composed of three subcomplexes. FqoF functions as the input device adjusted to the oxidation of reduced cofactor F420H2, thereby replacing subunits of the input module of complex I that are not present in A. fulgidus. The subunits FqoB, FqoCD and FqoI may form the membrane-associated module and transfer electrons to the membrane-integral module. It is most likely that the last subcomplex is composed of FqoA, FqoH, FqoJ, FqoK, FqoL, FqoM and FqoN. All subunits are highly hydrophobic and are probably involved in the reduction of a special menaquinone with a fully reduced isoprenoid side chain present in the cytoplasmic membrane of A. fulgidus.     

Purification of the NADP+:F420 oxidoreductase of Methanosphaera stadtmanae

Methanosphaera stadtmanae (DSM 3091) is a methanogen that requires H2 and CH3OH for methanogenesis. The organism does not possess an F420-dependent hydrogenase and only low levels of F420. It does however possess NADP+:F420 oxidoreductase activity. The NADP+:F420 oxidoreductase, the enzyme which catalyses the electron transfer between NADP+ and F420 in this organism, was purified and characterized. NAD+, NADH, FMN, and FAD could not be used as electron acceptors. Optimal pH for F420 reduction was 6.0, and 8.5 for NADP+ reduction. During the purification process, it was noted that precipitation with (NH4)2SO4 increased total activity 16-fold but reduced the stability of the enzyme. However, recombination of cell-free extracts with resuspended 65-90% (NH4)2SO4 pellet returned activity to near cell-free extract levels. Neither high salt or protease inhibitors were effective in stabilizing the activity of the partially purified enzyme. The purified enzyme from M. stadtmanae possessed a molecular weight of 148 kDa as determined by gel filtration chromatography and native-PAGE, consisting of alpha, beta, and gamma subunits of 60, 50, and 45 kDa, respectively, using SDS-PAGE. The Km values were 370 microM for NADP+, 142 microM for NADPH, 62.5 microM for F420, and 7.7 microM for F420H2. These values were different from the Km values observed in the cell-free extract.     

Purification and properties of the membrane-associated coenzyme F420-reducing hydrogenase from Methanobacterium formicicum.

The membrane-associated coenzyme F420-reducing hydrogenase of Methanobacterium formicicum was purified 87-fold to electrophoretic homogeneity. The enzyme contained alpha, beta, and gamma subunits (molecular weights of 43,000, 36,700, and 28,800, respectively) and formed aggregates (molecular weight, 1,020,000) of a coenzyme F420-active alpha 1 beta 1 gamma 1 trimer (molecular weight, 109,000). The hydrogenase contained 1 mol of flavin adenine dinucleotide (FAD), 1 mol of nickel, 12 to 14 mol of iron, and 11 mol of acid-labile sulfide per mol of the 109,000-molecular-weight species, but no selenium. The isoelectric point was 5.6. The amino acid sequence I-N3-P-N2-R-N1-EGH-N6-V (where N is any amino acid) was conserved in the N-termini of the alpha subunits of the F420-hydrogenases from M. formicicum and Methanobacterium thermoautotrophicum and of the largest subunits of nickel-containing hydrogenases from Desulfovibrio baculatus, Desulfovibrio gigas, and Rhodobacter capsulatus. The purified F420-hydrogenase required reductive reactivation before assay. FAD dissociated from the enzyme during reactivation unless potassium salts were present, yielding deflavoenzyme that was unable to reduce coenzyme F420. Maximal coenzyme F420-reducing activity was obtained at 55 degrees C and pH 7.0 to 7.5, and with 0.2 to 0.8 M KCl in the reaction mixture. The enzyme catalyzed H2 production at a rate threefold lower than that for H2 uptake and reduced coenzyme F420, methyl viologen, flavins, and 7,8-didemethyl-8-hydroxy-5-deazariboflavin. Specific antiserum inhibited the coenzyme F420-dependent but not the methyl viologen-dependent activity of the purified enzyme.     

Changes in concentrations of coenzyme F420 analogs during batch growth of Methanosarcina barkeri and Methanosarcina mazei

Coenzyme F420 has been assayed by high-performance liquid chromatography with fluorimetric detection; this permits quantification of individual coenzyme F420 analogs whilst avoiding the inclusion of interfering material. The total intracellular coenzyme F420 content of Methanosarcina barkeri MS cultivated on methanol and on H2-CO2 and of Methanosarcina mazei S-6 cultured on methanol remained relatively constant during batch growth. The most abundant analogs in M. barkeri were coenzymes F420-2 and F420-4, whilst in M. mazei coenzymes F420-2 and F420-3 predominated. Significant changes in the relative proportions of the coenzyme F420 analogs were noted during batch growth, with coenzymes F420-2 and F420-4 showing opposite responses to each other and the same being also true for coenzymes F420-3 and F420-5. This suggests that an enzyme responsible for transferring pairs of glutamic acid residues may be active. The degradation fragment FO was also detected in cells in late exponential and stationary phase. Coenzyme F420 analogs were present in the culture supernatant of both methanogens, in similar proportions to that in the cells, except for FO which was principally located in the supernatant.