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1.
Bruno Schwarzkopf Brigitte Reuke Andreas Kiener Adelbert Bacher 《Archives of microbiology》1990,153(3):259-263
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 相似文献
2.
The hydride carrier coenzyme F420 contains the unusual chromophore 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO). Microbes that generate F420 produce this FO moiety using a pyrimidine intermediate from riboflavin biosynthesis and the 4-hydroxyphenylpyruvate precursor of tyrosine. The fbiC gene, cloned from Mycobacterium smegmatis, encodes the bifunctional FO synthase. Expression of this protein in Escherichia coli caused the host cells to produce FO during growth, and activated cell-free extracts catalyze FO biosynthesis in vitro. FO synthase in the methanogenic euryarchaeon Methanocaldococcus jannaschii comprises two proteins encoded by cofG (MJ0446) and cofH (MJ1431). Both subunits were required for FO biosynthesis in vivo and in vitro. Cyanobacterial genomes encode homologs of both genes, which are used to produce the coenzyme for FO-dependent DNA photolyases. A molecular phylogeny of the paralogous cofG and cofH genes is consistent with the genes being vertically inherited within the euryarchaeal, cyanobacterial, and actinomycetal lineages. Ancestors of the cyanobacteria and actinomycetes must have acquired the two genes, which subsequently fused in actinomycetes. Both CofG and CofH have putative radical S-adenosylmethionine binding motifs, and pre-incubation with S-adenosylmethionine, Fe2+, sulfide, and dithionite stimulates FO production. Therefore a radical reaction mechanism is proposed for the biosynthesis of FO.Abbreviations
AdoMet (SAM)
S-adenosyl-l-methionine
-
Compound 6
5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione
-
FO
7,8-Didemethyl-8-hydroxy-5-deazariboflavin
-
HPP
4-Hydroxyphenylpyruvate 相似文献
3.
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. 相似文献
4.
L. Dudley Eirich Roberts S. Dugger 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,802(3):454-458
The F420-dependent NADP reductase of Methanobacterium thermoautotrophicum has been purified employing a combination of DEAE-cellulose ion-exchange chromatography, affinity chromatography with Blue Sepharose, Sephadex G-200 column chromatography and Red Sepharose affinity chromatography. The enzyme, which requires reduced F420 as an electron donor, has been purified over 3000-fold with a recovery of 65%. A molecular weight of 112000 was determined by Sephadex G-200 chromatography. A subunit molecular weight of 28 500 was determined by Sephadex G-200 chromatography. A subunit native enzyme is a tetramer. The optimal temperature for enzymatic activity was found to be 60°C with a pH optimum of 8.0. The NADP reductase had an apparent Km of 128 μMJ for reduced F420 and 40 μM for NADP. The enzyme was stable for at least 4 h at 65°C and pH 7.5. No loss of enzyme activity was detected when purified enzyme was stored aerobically in buffer containing 2-mercaptoethanol for 10 days at 4°C. Neither FMNH2 nor FADH2 could serve as electron donors; NAD was not utilized as electron acceptor. 相似文献
5.
Abstract Cell-free extracts of Methanobacterium thermoautotrophicum (strain ΔH) converted the 8-OH-5-deazaflavin coenzyme F420 to factor 390, a 8-adenylyl derivative (F420 -AMP). Activity was only observed upon exposure of the crude cell-free extract to oxygen. The ability to synthesize F390 was lost when crude cell-free extract was subsequently brought to an anaerobic reducing environment. The enzymatic reaction used ATP and oxidized coenzyme F420 as substrates and inorganic pyrophosphate was formed next to F390 . GTP could be used instead of ATP resulting in a guanylylated derivative. The crude cell-free extract showed K m values of 154 μM for coenzyme F420 and 2.4 mM for ATP. A partially purified enzyme preparation exhibited a K eq of 0.32. In accordance, coenzyme F420 and ATP could be synthesized from F390 and PPi by the reverse reaction. 相似文献
6.
Nocek B Evdokimova E Proudfoot M Kudritska M Grochowski LL White RH Savchenko A Yakunin AF Edwards A Joachimiak A 《Journal of molecular biology》2007,372(2):456-469
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. 相似文献
7.
Karl-Heinz Blotevogel Ulrich Fischer Margit Mocha Sigrid Jannsen 《Archives of microbiology》1985,142(3):211-217
An autotrophic moderately alkaliphilic and thermophilic nonmotile rod-shaped methanogen was isolated from a biogas plant. The isolate grows only on H2+CO2 and requires yeast extract. Growth optimum is at 60°C with a generation time of 4 h. In the absence of substrates complete lysis occurs. The pH range for growth is 7.5–8.5. Growth was also observed at pH values above 9.0. The DNA base composition is 38.8 mol% G+C. According to its physiological properties the nameMethanobacterium thermoalcaliphilum is proposed.Abbreviations G+C
Guanine+cytosine 相似文献
8.
Two serine racemases (I and II) were isolated from Streptomyces garyphalus. Serine racemase I (molecular weight 93,000) was purified to a single band in an analytical electrofocusing system. Serine racemase II (molecular weight 73,000) was partially purified. Both enzymes used pyridoxal-5-phosphate as cofactor. Besides serine the enzymes utilized alanine as substrate but no other amino acid tested. The K
m values of l-alanine and l-serine for enzyme I were 111 mM and 35 mM respectively. Enzyme I was not inhibited by d-cycloserine but by hydroxylamine. Both substances inhibited enzyme II. The serine racemases may be involved in the biosynthesis of d-cycloserine in S. garyphalus. 相似文献
9.
Marie-Louise Svensson Kristoffer Valeria Sten Gatenbeck 《Archives of microbiology》1981,129(3):210-212
It was found that hydroxyurea, l-arginine and l-citrulline respectively significantly stimulated the formation of d-cycloserine in Streptomyces garyphalus. The formation of [14C]-hydroxyurea by washed cells was demonstrated after incubation with l-[guanido-14C]-arginine and l-[ureido-14C]-citrulline. The 15N of H2NCO15NHOH was incorporated to 40% in d-cycloserine. The mass spectrum as well as the 15N NMR spectrum of labelled N,2-dicarbobenzyloxy-d-cycloserine derived from [15N]-hydroxyurea showed that hydroxyurea was the source of the heterocyclic nitrogen in the biosynthesis of d-cycloserine. 相似文献
10.
Jasper Kunow Beatrix Schwörer Karl O. Stetter Rudolf K. Thauer 《Archives of microbiology》1993,160(3):199-205
Archaeoglobus fulgidus, a sulfate-reducing Archaeon with a growth temperature optimum of 83°C, uses the 5-deazaflavin coenzyme F420 rather than pyridine nucleotides in catabolic redox processes. The organism does, however, require reduced pyridine nuclcotides for biosynthetic purposes. We describe here that the Archaeon contains a coenzyme F420-dependent NADP reductase which links anabolism to catabolism. The highly thermostable enzyme was purfied 3600-fold by affinity chromatography to apparent homogeneity in a 60% yield. The native enzyme with an apparent molecular mass of 55 kDa was composed of only one type of subunit of apparent molecular mass of 28 kDa. Spectroscopic analysis of the enzyme did not reveal the presence of any chromophoric prosthetic group. The purified enzyme catalyzed the reversible reduction of NADP (apparent K
M 40 M) with reduced F420 (apparent K
M 20M) with a specific activity of 660 U/mg (apparent V
max) at pH 8.0 (pH optimum) and 80°C (temperature optimum). It was specific for both coenzyme F420 and NADP. Sterochemical investigations showed that the F420-dependent NADP reductase was Si face specific with respect to C5 of F420 and Si face specific with respect to C4 of NADP.Abbreviations F420
coenzyme F420
- F420H2
1,5-dihydrocoenzyme F420
- H4MPT
tetrahydromethanopterin
- CH=H4MPT
N5, N10-methylenetetrahydromethanopterin
- MFR
methanofuran
- HPLC
high performance liquid chromatography
- methylene-H4MPT dehydrogenase
N5, N10-methylenetetrahydromethanopterin dehydrogenase
- 1 U =
1 mol/min 相似文献
11.
Andreas R. Klein Jürgen Koch Karl O. Stetter Rudolf K. Thauer 《Archives of microbiology》1993,160(3):186-192
It was recently reported that the extreme thermophile Methanopyrus kandleri contains only a H2-forming N
5, N
10-methylenetetrahydromethanopterin dehydrogenase which uses protons as electron acceptor. We describe here the presence in this Archaeon of a second N
5,N
10-methylenetetrahydromethanopterin dehydrogenase which is coenzyme F420-dependent. This enzyme was purified and characterized. The enzyme was colourless, had an apparent molecular mass of 300 kDa, an isoelectric point of 3.7±0.2 and was composed of only one type of subunit of apparent molecular mass of 36 kDa. The enzyme activity increased to an optimum with increasing salt concentrations. Optimal salt concentrations were e.g. 2 M (NH4)2SO4, 2 M Na2HPO4, 1.5 M K2HPO4, and 2 M NaCl. In the absence of salts the enzyme exhibited almost no activity. The salts affected mainly the V
max rather than the K
m of the enzyme. The catalytic mechanism of the dehydrogenase was determined to be of the ternary complex type, in agreement with the finding that the enzyme lacked a chromophoric prosthetic group. In the presence of M (NH4)2SO4 the V
max was 4000 U/mg (k
cat=2400 s-1) and the K
m for N
5,N
10-methylenetetrahydromethanopterin and for coenzyme F420 were 80 M and 20 M, respectively. The enzyme was relatively heat-stable and lost no activity when incubated anaerobically in 50 mM K2HPO4 at 90°C for one hour. The N-terminal amino acid sequence was found to be similar to that of the F420-dependent N
5, N
10-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Archaeoglobus fulgidus.Abbreviations H4MPT
tetrahydromethanopterin
- F420
coenzyme F420
- CH2=H4MPT
N
5,N
10-methylenetrahydromethanopterin
- CHH4MPT+
N
5,N
10-methenyltetrahydromethanopterin
- methylene-H4MPT dehydrogenase
N
5,N
10-methylenetetrahydromethanopterin dehydrogenase
- Mops
N-morpholinopropane sulfonic acid
- Tricine
N-[Tris(hydroxymethyl)-methyl]glycine
- 1 U =
1 mol/min 相似文献
12.
Methanobacterium thermoautotrophicum, growing on medium supplemented with 2 mol 63NiCl2/l, was found to take up 1.2 mol 63Ni per g cells (dry weight). More than 70% of the radioisotope was incorporated into a compound, which dissociated from the protein fraction after heat treatment, was soluble in 70% acetone, and could be purified by chromatography on QAE-Sephadex A-25, Sephadex G-25, and DEAE cellulose. The purified 63Ni labelled compound had an absorption spectrum and properties identical to those of factor F
430 and is therefore considered to be identical with factor F
430.Factor F
430, a compound of molecular weight higher than 1000 with an absorbance maximum at 430 nm, has recently been purified from Methanobacterium thermoautotrophicum (Gunsalus and Wolfe, 1978). The structure and function of this compound are not yet known. 相似文献
13.
Gabriele Diekert Hans-Harald Gilles Rolf Jaenchen Rudolf K. Thauer 《Archives of microbiology》1980,128(2):256-262
Factors F430 from methanogenic bacteria have recently been shown to contain nickel and it has been speculated that they may have a nickel tetrapyrrole structure. This assumption was tested by determining whether succinate is incorporated by growing Methanobacterium thermoautotrophicum into three factors F430. Succinate is assimilated by Methanobacterium thermoautotrophicum into the amino acids glutamate, arginine and proline and into tetrapyrroles rather than other cell components. It was found that per mol nickel 8–9 mol of succinate were incorporated into the three factors F430 which is the amount predicted for a tetrapyrrole structure. Since the three factors F430 only contained significant amounts of glutamate rather than arginine or proline, the incorporation data suggest that factors F430 are nickel tetrapyrrole compounds. Spectral properties of the three factors F430, apparent molecular weights, and the absence of phosphor in these compounds are also described. 相似文献
14.
S. Rospert J. Breitung K. Ma B. Schwörer C. Zirngibl R. K. Thauer D. Linder R. Huber K. O. Stetter 《Archives of microbiology》1991,156(1):49-55
Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H2 and CO2 and that shows no close phylogenetic relationship to any methanogen known so far. Methyl-coenzyme M reductase, the enzyme catalyzing the methane forming step in the energy metabolism of methanogens, was purified from this hyperthermophile. The yellow protein with an absorption maximum at 425 nm was found to be similar to the methyl-coenzyme M reductase from other methanogenic bacteria in that it was composed each of two -, - and -subunits and that it contained the nickel porphinoid coenzyme F430 as prosthetic group. The purified reductase was inactive. The N-terminal amino acid sequence of the -subunit was determined. A comparison with the N-terminal sequences of the -subunit of methyl-coenzyme M reductases from other methanogenic bacteria revealed a high degree of similarity.Besides methyl-coenzyme M reductase cell extracts of M. kandleri were shown to contain the following enzyme activities involved in methanogenesis from CO2 (apparent Vmax at 65°C): formylmethanofuran dehydrogenase, 0.3 U/mg protein; formyl-methanofuran: tetrahydromethanopterin formyltransferase, 13 U/mg; N
5,N10-methenyltetrahydromethanopterin cyclohydrolase, 14 U/mg; N
5,N10-methylenetetrahydromethanopterin dehydrogenase (H2-forming), 33 U/mg; N
5,N10-methylenetetrahydromethanopterin reductase (coenzyme F420 dependent), 4 U/mg; heterodisulfide reductase, 2 U/mg; coenzyme F420-reducing hydrogenase, 0.01 U/mg; and methylviologen-reducing hydrogenase, 2.5 U/mg. Apparent Km values for these enzymes and the effect of salts on their activities were determined.The coenzyme F420 present in M. kandleri was identified as coenzyme F420-2 with 2 -glutamyl residues.Abbreviations H–S-CoM
coenzyme M
- CH3–S-CoM
methylcoenzyme M
- H–S-HTP
7-mercaptoheptanoylthreonine phosphate
- MFR
methanofuran
- CHO-MFR
formyl-MFR
- H4MPT
tetrahydromethanopterin
- CHO–H4MPT
N
5-formyl-H4MPT
- CH=H4MPT+
N
5,N10-methenyl-H4MPT
- CH2=H4MPT
N
5,N10-methylene-H4MPT
- CH3–H4MPT
N
5-methyl-H4MPT
- F420
coenzyme F420
- 1 U=
1 mol/min 相似文献
15.
We measured F420-dependent N5,N10-methylenetetrahydro-methanopterin dehydrogenase, N5, N10-methenyltetrahydro-methanopterin cyclohydrolase, and F420-reducing hydrogenase levels in Methanosarcina barkeri grown on various substrates. Variation in dehydrogenase levels during growth on a specific substrate was usually <3-fold, and much less for cyclohydrolase. H2–CO2-, methanol-, and H2–CO2+ methanol-grown cells had roughly equivalent levels of dehydrogenase and cyclohydrolase. In acetate-grown cells cyclohydrolase level was lowered 2 to 3-fold and dehydrogenase 10 to 80-fold; this was not due to repression by acetate, since, if cultures growing on acetate were supplemented with methanol or H2–CO2, dehydrogenase levels increased 14 to 19-fold, and cyclohydrolase levels by 3 to 4-fold. Compared to H2–CO2- or methanol-grown cells, acetate-or H2–CO2 + methanol-grown cells had lower levels of and less growth phase-dependent variation in hydrogenase activity. Our data are consistent with the following hypotheses: 1. M. barkeri oxidizes methanol via a portion of the CO2-reduction pathway operated in the reverse direction. 2. When steps from CO2 to CH3-S-CoM in the CO2-reduction pathway (in either direction) are not used for methanogenesis, hydrogenase activity is lowered.Abbreviations MF
methanofuran
- H4MPT
5,6,7,8-tetrahydromethanopterin
- HS-HTP
7-mercaptoheptanoylthreonine phosphate
- CoM-S-S-HTP
heterodisulfide of HS-CoM and HS-HTP
- F420
coenzyme F420 (a 7,8-didemethyl-8-hydroxy-5-deaza-riboflavin derivative)
- H2F420
reduced coenzyme F420
- HC+=H4MPT
N5,N10-methenyl-H4MPT
- H2C=H4MPT
N5,N10-methylene-H4MPT
- H3C=H4MPT
N5-methyl-H4MPT
- BES
2-bromoethanesulfonic acid 相似文献
16.
S. F. Baron D. S. Williams H. D. May P. S. Patel H. C. Aldrich J. G. Ferry 《Archives of microbiology》1989,151(4):307-313
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 相似文献
17.
Methanobacterium thermoautotrophicum was found to require sodium for growth and for CO2 reduction to methane. The dependence of the rate of growth and methane formation on the sodium concentration was hyperbolic with an apparent K
s for sodium of approximately 1 mM. The findings indicate that sodium has a specific function in the energy metabolism of this bacterium. 相似文献
18.
The activities of formylmethanofuran dehydrogenase, methylenetetrahydromethanopterin dehydrogenase, methylenetetrahydromethanopterin
reductase, and heterodisulfide reductase were tested in cell extracts of 10 different methanogenic bacteria grown on H2/CO2 or on other methanogenic substrates. The four activities were found in all the organisms investigated: Methanobacterium thermoautotrophicum,M. wolfei, Methanobrevibacter arboriphilus, Methanosphaera stadtmanae, Methanosarcina
barkeri (strains Fusaro and MS), Methanothrix soehngenii, Methanospirillum hungatei, Methanogenium organophilum, and Methanococcus voltae. Cell extracts of H2/CO2 grown M. barkeri and of methanol grown M. barkeri showed the same specific activities suggesting that the four enzymes are of equal importance in CO2 reduction to methane and in methanol disproportionation to CO2 and CH4. In contrast, cell extracts of acetate grown M. barkeri exhibited much lower activities of formylmethanofuran dehydrogenase and methylenetetrahydromethanopterin dehydrogenase suggesting
that these two enzymes are not involved in methanogenesis from acetate. In M. stadtmanae, which grows on H2 and methanol, only heterodisulfide reductase was detected in activities sufficient to account for the in vivo methane formation rate. This finding is consistent with the view that the three other oxidoreductases are not required for
methanol reduction to methane with H2. 相似文献
19.
Kin Sing Lam Jacqueline Mattei Salvatore Forenza 《Journal of industrial microbiology & biotechnology》1989,4(2):105-108
Summary A new antitumor antibiotic named rebeccamycin was isolated from fermentations of an actinomycete,Saccharothrix aerocolonigenes. A defined medium was developed to study the regulation of synthesis of rebeccamycin byS. aerocolonigenes. In glucose medium formation of rebeccamycin was detected only after glucose was depleted. Examination of eleven different carbon sources revealed that carbon catabolite regulation is a major control mechanism for rebeccamycin production. 相似文献
20.
Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H2 and CO2 and that shows no close phylogenetic relationship to any methanogens known so far. N
5
N
10
-Methylenetetrahydromethanopterin reductase, an enzyme involved in methanogenesis from CO2, was purified from this hyperthermophile. The apparent molecular mass of the native enzyme was found to be 300 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only one polypeptide of apparent molecular mass 38 kDa. The ultraviolet/visible spectrum of the enzyme was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was specific for reduced coenzyme F420 as electron donor; NADH, NADPH or reduced dyes could not substitute for the 5-deazaflavin. The catalytic mechanism was found to be of the ternary complex type as deduced from initial velocity plots. V
max at 65°C and pH 6.8 was 435 U/mg (kcat=275 s-1) and the K
m for methylenetetrahydro-methanopterin and for reduced F420 were 6 M and 4 M, respectively. From Arrhenius plots an activation energy of 34 kJ/mol was determined. The Q
10 between 40°C and 90°C was 1.5.The reductase activity was found to be stimulated over 100-fold by sulfate and by phosphate. Maximal stimulation (100-fold) was observed at a sulfate concentration of 2.2 M and at a phosphate concentration of 2.5 M. Sodium-, potassium-, and ammonium salts of these anions were equally effective. Chloride, however, could not substitute for sulfate or phosphate in stimulating the enzyme activity.The thermostability of the reductase was found to be very low in the absence of salts. In their presence, however, the reductase was highly thermostable. Salt concentrations between 0.1 M and 1.5 M were required for maximal stability. Potassium salts proved more effective than ammonium salts, and the latter more effective than sodium salts in stabilizing the enzyme activity. The anion was of less importance.The N-terminal amino acid sequence of the reductase from M. kandleri was determined and compared with that of the enzyme from Methanobacterium thermoautotrophicum and Methanosarcina barkeri. Significant similarity was found.Abbreviations H4MPT
tetrahydromethanopterin
- CH2=H4MPT
N
5
,N
10
-methylene-H4MPT
- CH3-H4MPT
N
5-methyl-H4MPT
- CHH4MPT+
N
5
,N
10
-methenyl-H4MPT
- F420
coenzyme F420; 1 U=1 mol/min 相似文献