Distribution of charged pterins in nonmethanogenic archaebacteria

Pterin content was surveyed in nine species of nonmethanogenic archaebacteria comprised of six species of halobacteria, two species of Sulfolobus, and one specie of Thermoplasma. The results indicated the presence of the sulfate-containing dimeric pterin sulfohalopterin-2 in three species of halobacteria, Halobacterium marismortui, halobacterial strain GN-1, and Halobacterium volcanii. The phosphate-containing dimeric pterin phosphohalopterin-1 was present in three other species of halobacteria, Halobacterium salinarium, Halobacterium halobium, and Halococcus morrhuae. Evidence is presented that these halopterins exist in the halobacteria as tetrahydropterin derivatives. A positively charged monomeric pterin, solfapterin (erythro-neopterin-3-D-2-deoxy-2-aminoglucopyranoside), was found to be present in Sulfolobus solfataricus and a negatively charged pterin was induced when S. solfataricus was grown in a medium containing homogentisic acid (2,5-dihydroxyphenylacetic acid). This negatively charged pterin was not present when the cells were grown in the normal medium. Another positively charged pterin found in Thermoplasma was determined to be different from solfapterin based on its paper electrophoresis properties. Uncharged pterins were also identified in all of these bacteria, however, no attempt was made to elucidate the structure of these uncharged pterins. Pterin content was found to vary in the different bacteria and to depend on the conditions under which the cells were grown.     

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.     

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     

Analysis and characterization of the folates in the nonmethanogenic archaebacteria.

A detailed analysis of the folate coenzymes in the nonmethanogenic archaebacteria has been performed. By using the Lactobacillus casei microbiological assay for folates, the levels of folates in Sulfolobus solfataricus and Sulfolobus acidocaldarius were found to be 3.7 and 8.3 ng/g (dry weight) of cells, respectively, compared with 88,000 and 28,000 ng/g (dry weight) of cells in Halobacterium halobium and Halobacterium strain GN-1, respectively. The levels of folates found in the Sulfolobus spp. were approximately 100 times less than those found in the typical eubacterium, whereas the levels in the halobacteria were approximately 10 times higher. The folate in Sulfolobus solfataricus was shown to consist of only 5-formyltetrahydropteroylglutamate, and the folate in Halobacterium strain GN-1 was shown to consist of only pteroyldiglutamate. The low folate levels in the Sulfolobus spp. are the same as those found in the methanogenic bacteria, suggesting that another C1 carrier may function in these cells.     

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.     

Levels of coenzyme F420, coenzyme M, hydrogenase, and methylcoenzyme M methylreductase in acetate-grown Methanosarcina.

Methanosarcina barkeri strain 227 maintained on an acetate medium for 2 years was found to possess hydrogenase, methylcoenzyme M methylreductase, coenzyme F420, and coenzyme M. The levels of these constituents in acetate-grown cells were similar to those found in cells of the same strain grown on methanol or hydrogen and carbon dioxide.     

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.     

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.     

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.     

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.     

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.     

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     

Function of methanofuran,tetrahydromethanopterin, and coenzyme F420 in Archaeoglobus fulgidus

Archaeoglobus fulgidus is an extremely thermophilic archaebacterium that can grow at the expense of lactate oxidation with sulfate to CO₂ and H₂S. The organism contains coenzyme F₄₂₀, tetrahydromethanopterin, and methanofuran which are coenzymes previously thought to be unique for methanogenic bacteria. We report here that the bacterium contains methylenetetrahydromethanopterin: F₄₂₀ oxidoreductase (20 U/mg), methenyltetrahydromethanopterin cyclohydrolase (0.9 U/mg), formyltetrahydromethanopterin: methanofuran formyltransferase (4.4 U/mg), and formylmethanofuran: benzyl viologen oxidoreductase (35 mU/mg). Besides these enzymes carbon monoxide: methyl viologen oxidoreductase (5 U/mg), pyruvate: methyl viologen oxidoreductase (0.7 U/mg), and membranebound lactate: dimethylnaphthoquinone oxidoreductase (0.1 U/mg) were found. 2-Oxoglutarate dehydrogenase, which is a key enzyme of the citric acid cycle, was not detectable. From the enzyme outfit it is concluded that in A. fulgidus lactate is oxidized to CO₂ via a modified acetyl-CoA/carbon monoxide dehydrogenase pathway involving C₁-intermediates otherwise only used by methanogenic bacteria.Non-standard abbreviations APS adenosine 5-phosphosulfate - BV benzyl viologen - DCPIP 2,6-dichlorophenolindophenol - DMN 2,3-dimethyl-1,4-naphthoquinone - DTT DL-1,4-dithiothreitol - H₄F tetrahydrofolate - H₄MPT tetrahydromethanopterin - CH₂ H₄MPT, methylene-H₄MPT - CH H₄MPT, methenyl-H₄MPT - Mes morpholinoethane sulfonic acid - MFR methanofuran - Mops morpholinopropane sulfonic acid - MV methyl viologen - Tricine N-tris(hydroxymethyl)-methylglycine - U mol product formed per min     

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.     

Effect of temperature on the spectral properties of coenzyme F420 and related compounds.

The uv-visible spectra of 7,8-didemethyl-8-hydroxy-5-deazaflavin-5'-phosphoryllactyl glutamate (coenzyme F420), a naturally occurring 5-deazaflavin derivative, in three different buffers changed with a rise in temperature; the effect on the extinction coefficient at 420 nm (epsilon 420) was as follows: In phosphate-buffered solutions at pH less than 7.5, the epsilon 420 increased (at pH 5.0 for a temperature shift from 15 to 60 degrees C, delta epsilon 420 was +87%), but between pH 7.5 and 8, epsilon 420 changed very little. At pH greater than 8.0 in phosphate- or borate-buffered solutions, epsilon 420 decreased slightly. In morpholineethanesulfonic acid (Mes)-buffered F420 solutions at pH 5 and 5.5, epsilon 420 changed very little, whereas at pH 6-8, the epsilon 420 decreased. Absorbance of F420 at 401 nm in phosphate buffer at pH 5 to 9 was not significantly affected by temperature. Changes in epsilon 420 due to temperature change corresponded to changes in the pKa of 8-OH of the deazaflavin molecule; studies with adenylated F420 showed that the 8-OH of F420 was responsible for these changes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Levels of coenzyme F420, coenzyme M, hydrogenase, and methylcoenzyme M methylreductase in acetate-grown Methanosarcina

Methanosarcina barkeri strain 227 maintained on an acetate medium for 2 years was found to possess hydrogenase, methylcoenzyme M methylreductase, coenzyme F420, and coenzyme M. The levels of these constituents in acetate-grown cells were similar to those found in cells of the same strain grown on methanol or hydrogen and carbon dioxide.     

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.     

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.     

Quantification of coenzyme F420 analogues from methanogenic bacteria by HPLC and fluorimetry

Summary A quantitative assay for analogues of coenzyme F₄₂₀ is presented. The assay combines separation of the coenzymes by binary reversed-phase high performance liquid chromatography with detection by fluorescence, yielding high specificity and sensitivity. Quantification is by calibration with a coenzyme F₄₂₀ standard or by employing coenzyme F₄₂₀ fragments as internal standards.