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1.
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
2.
The dehydrogenation of N
5,N
10-methylenetetrahydromethanopterin (CH2=H4MPT) to N
5,N
10-methenyltetrahydromethanopterin (CH≡H4MPT+) is an intermediate step in the oxidation of methanol to CO2 in Methanosarcina barkeri. The reaction is catalyzed by CH2=H4MPT dehydrogenase, which was found to be specific for coenzyme F420 as electron acceptor; neither NAD, NADP nor viologen dyes could substitute for the 5-deazaflavin. The dehydrogenase was anaerobically
purified almost 90-fold to apparent homogeneity in a 32% yield by anion exchange chromatography on DEAE Sepharose and Mono
Q HR, and by affinity chromatography on Blue Sepharose. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed
only one protein band with an apparent mass of 31 kDa. The apparent molecular mass of the native enzyme determined by polyacrylamide
gradient gel electrophoresis was 240 kDa. The ultraviolet/visible spectrum of the purified enzyme was almost identical to
that of albumin suggesting the absence of a chromophoric prosthetic group. Reciprocal plots of the enzyme activity versus
the substrate concentrations were linear: the apparent K
m for CH2=H4MPT and for coenzyme F420 were found to be 6 μM and 25 μM, respectively. Vmax was 4,000 μmol min-1·mg-1 protein (kcat=2,066 s-1) at pH 6 (the pH optimum) and 37°C. The Arrhenius activation energy was 40 kJ/mol. The N-terminal amino acid sequence was found to be 50% identical with that of the F420-dependent CH2=H4MPT dehydrogenase isolated from H2/CO2 grown Methanobacterium thermoautotrophicum. 相似文献
3.
Methylene-H4MPT reductase was found to be present in Archaeoglobus fulgidus in a specific activity of 1 U/mg. The reductase was purified 410-fold. The native enzyme showed an apparent molecular mass of approximately 200 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only 1 polypeptide of apparent molecular mass 35 kDa. The ultraviolet/visible spectrum of the reductase was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was dependent on reduced coenzyme F420 as electron donor. Neither NADH, NADPH, nor reduced viologen dyes could substitute for the reduced deazaflavin. From reciprocal plots, which showed an intersecting patter, a K
m for methylene-H4MPT of 16 M, a K
m for F420H2 of 4 M, and a V
max of 450 U/mg (Kcat=265 s-1) were obtained. The enzyme was found to be rapidly inactivated when incubated at 80°C in 100 mM Tris/HCl pH 7. The rate of inactivation, however, decreased to essentially zero in the presence of either F420 (0.2 mM), methylene-H4MPT (0.2 mM), albumin (1 mg/ml), or KCl (0.5 M). The N-terminal amino acid sequence was determined and found to be similar to that of methylene-H4MPT reductase (F420-dependent) from the methanogens Methanobacterium thermoautotrophicum, Methanosarcina barkeri, and Methanopyrus kandleri. The purification and some properties of formylmethanofuran dehydrogenase from A. fulgidus are also described.Abbreviations H4MPT
tetrahydromethanopterin
- CH2=H4MPT
N
5,N
10-methylene-H4MPT
- CH3–H4MPT
N
5-methyl-H4MPT
- CHH4MPT
methenyl-H4MPT
- F420
coenzyme F420
- MFR
methanofuran
- CHO-MFR
formyl-MFR
- 1 U
1 mol/min 相似文献
4.
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
5.
A. R. Klein J. Breitung D. Linder K. O. Stetter R. K. Thauer 《Archives of microbiology》1993,159(3):213-219
Archaeoglobus fulgidus and Methanopyrus kandleri are both extremely thermophilic Archaea with a growth temperature optimum at 83°C and 98°C, respectively. Both Archaea contain an active N
5,N
10-methenyltetrahydromethanopterin cyclohydrolase. The enzyme from M. kandleri has recently been characterized. We describe here the purification and properties of the enzyme from A. fulgidus.The cyclohydrolase from A. fulgidus was purified 180-fold to apparent homogeneity and its properties were compared with those recently published for the cyclohydrolase from M. kandleri. The two cytoplasmic enzymes were found to have very similar molecular and catalytic properties. They differed, however, significantly with respect of the effect of K2HPO4 and of other salts on the activity and the stability. The cyclohydrolase from A. fulgidus required relatively high concentrations of K2HPO4 (1 M) for optimal thermostability at 90°C but did not require salts for activity. Vice versa, the enzyme from M. kandleri was dependent on high K2HPO4 concentrations (1.5 M) for optimal activity but not for thermostability. Thus the activity and structural stability of the two thermophilic enzymes depend in a completely different way on the concentration of inorganic salts. The molecular basis for these differences are discussed.Abbreviations H4MPT
tetrahydromethanopterin
- MFR
methanofuran
- CH3–H4MPT
N
5-methyl-H4MPT
- CH2=H4MPT
N
5,N
10-methylene-H4MPT
- CH2H4MPT
N
5,N
10-methenyl-H4MPT
- CHO–H4MPT
N
5 formyl-H4MPT
- CHO-MFR
formyl-MFR
- cyclohydrolase
N
5,N
10-methenyltetrahydromethanopterin cyclohydrolase
- MOPS
3-(N-morpholino) propane sulfonic acid
- TRICINE
N-tris (hydroxymethyl) methyl glycine
- 1 U=1
mol/min 相似文献
6.
A bacterium tentatively classified as Arthrobacter strain Py1 being capable to degrade pyrrole-2-carboxylate as only source of carbon, nitrogen, and energy was isolated from soil. In contrast to many other N-heterocyclic compounds, growth of the isolate on pyrrole-2-carboxylate was not affected by molybdate or its specific inhibitor tungstate, indicating a molybdoenzyme-independent breakdown. The latter was initiated by a hydroxylation reaction catalyzed by a pyrrole-2-carboxylate oxygenase, which also exhibited an NADH-cytochrome c reductase activity. The pyrrole-2-carboxylate oxygenase reaction as examined in cell extracts depended on NADH, FAD, and pyrrole-2-carboxylate; the apparent K
m values were 44, 6, and 43 M, respectively. A degradation pathway for pyrrole-2-carboxylate is proposed which involves 5-hydroxy-pyrrole-2-carboxylate and 2-oxoglutarate. 相似文献
7.
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
8.
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
9.
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
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.
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
12.
Hagemeier CH Shima S Thauer RK Bourenkov G Bartunik HD Ermler U 《Journal of molecular biology》2003,332(5):1047-1057
The fourth reaction step of CO(2)-reduction to methane in methanogenic archaea is catalyzed by coenzyme F(420)-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd). We have structurally characterized this enzyme in the selenomethionine-labelled form from the hyperthermophilic methanogenic archaeon Methanopyrus kandleri at 1.54A resolution using the single wavelength anomalous dispersion method for phase determination. Mtd was found to be a homohexameric protein complex that is organized as a trimer of dimers. The fold of the individual subunits is composed of two domains: a larger alpha,beta domain and a smaller helix bundle domain with a short C-terminal beta-sheet segment. In the homohexamer the alpha,beta domains are positioned at the outside of the enzyme, whereas, the helix bundle domains assemble towards the inside to form an unusual quarternary structure with a 12-helix bundle around a 3-fold axis. No structural similarities are detectable to other enzymes with F(420) and/or substituted tetrahydropterins as substrates. The substrate binding sites of F(420) and methylenetetrahydromethanopterin are most likely embedded into a crevice between the domains of one subunit, their isoalloxazine and tetrahydropterin rings being placed inside a pocket formed by this crevice and a loop segment of the adjacent monomer of the dimer. Mtd revealed the highest stability at low salt concentrations of all structurally characterized enzymes from M.kandleri. This finding might be due to the compact quaternary structure that buries 36% of the monomer surface and to the large number of ion pairs. 相似文献
13.
B. Schwörer J. Breitung A. R. Klein K. O. Stetter R. K. Thauer 《Archives of microbiology》1993,159(3):225-232
The sulfate-reducing Archaeoglobus fulgidus contains a number of enzymes previously thought to be unique for methanogenic Archaea. The purification and properties of two of these enzymes, of formylmethanofuran: tetrahydromethanopterin formyltransferase and of N
5,N
10-methylenetetrahydromethanopterin dehydrogenase (coenzyme F420 dependent) are described here. A comparison of the N-terminal amino acid sequences and of other molecular properties with those of the respective enzymes from three methanogenic Archaea revealed a high degree of similarity.Abbreviations H4MPT
tetrahydromethanopterin
- F420
coenzyme
- F420
formyltransferase, formylmethanofuran: tetrahydromethanopterin formyltransferase
- methylene-H4MPT dehydrogenase
N
5,N
10-methylenetetrahydromethanopterin dehydrogenase
- methylene-H4MPT recductase
N
5,N
10-methylenetetrahydromethanopterin reductase
- cyclohydrolase
N
5,N
10-methenyltetrahydromethanopterin cyclohydrolase
- APS
adenosine 5-phosphosulfate
- MOPS
3-(N-morpholino) propane sulfonic acid
- TRICINE
N-tris(hydroxymethyl)methylglycine
- MES
morpholinoethanesulfonic acid
- 1 U
1 mol/min 相似文献
14.
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. 相似文献
15.
The activity of purified N
5,N
10-methenyltetrahydromethanopterin cyclohydrolase from Methanopyrus kandleri was found to increase up to 200-fold when potassium phosphate was added in high concentrations (1.5 M) to the assay. A 200-fold stimulation was also observed with sodium phosphate (1 M) and sodium sulfate (1 M) whereas stimulation by potassium sulfate (0.8 M), ammonium sulfate (1.5 M), potassium chloride (2.5 M), and sodium chloride (2 M) was maximal 100-fold. A detailed kinetic analysis of the effect of potassium phosphate revealed that this salt exerted its stimulatory effect by decreasing the K
m for N
5,N
10-methenyltetrahydromethanopterin from 2 mM to 40 M and by increasing the V
max from 2000 U/mg (kcat=1385 s-1) to 13300 U/mg (kcat=9200 s-1). Besides increasing the catalytic efficiency (kcat/K
m) salts were found to protect the cyclohydrolase from heat inactivation. For maximal thermostability much lower concentrations (0.1 M) of salts were required than for maximal activity.Abbreviations H4MPT
tetrahydromethanopterin
-
N
5,N
10-methenyl-H4MPT
- CHO-H4MPT
N
5-formyl-H4-MPT
- CH2=H4MPT
N
5,N
10-methylene-H4MPT
- CH3–H4-MPT
N
5-methyl-H4MPT
- MOPS
-N-morpholinopropane sulfonic acid
- TRICINE
N-[Tris(hydroxymethyl)-methyl]glycine
- 1 U =
1 mol/min 相似文献
16.
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 相似文献
17.
J. T. Keltjens J. M. H. Hermans G. J. F. A. Rijsdijk C. Van der Drift G. D. Vogels 《Antonie van Leeuwenhoek》1988,54(3):207-220
F430 is the prosthetic group of the methylcoenzyme M reductase of methanogenic bacteria. The compound isolated from Methanosarcina barkeri appears to be identical to the one obtained from the only distinctly related Methanobacterium thermoautotrophicum. F430 is thermolabile and in the presence of acetonitrile or C10
in4
sup-
two epimerization products are obtained upon heating; in the absence of these compounds F430 is oxidized to 12, 13-didehydro-F430. The latter is stereoselectively reduced under H2 atmosphere to F430 by cell-free extracts of M. barkeri or M. thermoautotrophicum. H2 may be replaced by the reduced methanogenic electron carrier coenzyme F420.Abbreviations CH3S-CoM
methylcoenzyme M, 2-methylthioethanesulfonic acid
- HS-CoM
coenzyme M, 2-mercaptoethanesulfonic acid
- F430
Ni(II) tetrahydro-(12, 13)-corphin with a uroporphinoid (III) ligand skeleton
- 13-epi-F430 and 12,13-di-epi-F430
the 12, 13- and 12, 13-derivatives of F430
- 12, 13-didehydro-F430
F430 oxidized at C-12 and C-13
- coenzyme F420
7,8-didemethyl-8-hydroxy-5-deazaflavin derivative
- coenzyme F420H2
reduced coenzyme F420
- MV+
methylviologen semiquinone
- HPLC
high-performance liquid chromatography 相似文献
18.
Acetate-grown cells of Methanosarcina barkeri MS were found to form methane from H2:CO2 at the same rate as hydrogen-grown cells. Cells grown on acetate had similar levels of soluble F420-reactive hydrogenase I, and higher levels of cytochrome-linked hydrogenase II compared to hydrogen-grown cells. The hydrogenase I and II activities in the crude extract of acetate-grown cells were separated by differential binding properties to an immobilized Cu2+ column. Hydrogenase II did not react with ferredoxin or F420, whereas hydrogenase I coupled to both ferredoxin and F420. A reconstituted soluble protein system composed of purified CO dehydrogenase, F420-reactive hydrogenase I fraction, and ferredoxin produced H2 from CO oxidation at a rate of 2.5 nmol/min · mg protein. Membrane-bound hydrogenase II coupled H2 consumption to the reduction of CoM-S-S-HTP and the synthesis of ATP. The differential function of hydrogenase I and II is ascribed to ferredoxin-linked hydrogen production from CO and cytochrome b-linked H2 consumption coupled to methanogenesis and ATP synthesis, respectively. 相似文献
19.
Gudrun C. Hartmann Andreas R. Klein Monica Linder Rudolf K. Thauer 《Archives of microbiology》1996,165(3):187-193
H2-FormingN
5,N10-methylenetetrahydromethanopterin dehydrogenase (Hmd) is a novel type of hydrogenase found in methanogenic Achaea that contains neither nickel nor iron-sulfur clusters. The enzyme has previously been characterized fromMethanobacterium thermoautotrophicum and fromMethanopyrus kandleri. We report here on the purification and properties of the enzyme fromMethanococcus thermolithotrophicus. Thehmd gene was cloned and sequenced. The results indicate that the enzyme fromMc. thermolithotrophicus is functionally and structurally closely related to the H2-forming methylene tetrahydromethanopterin dehydrogenase fromMb. thermoautotrophicum andMp. kandleri. From amino acid sequence comparisons of the three enzymes, a phylogenetic tree was deduced that shows branching orders similar to those derived from sequence comparisons of the 16S rRNA of the orders Methanococcales, Methanobacteriales, and Methanopyrales.Abbreviations
H
2
Forming dehydrogenase orHmd
- H2-FormingN
5,N10
methylene tetrahydromethanopterin dehydrogenase
-
H
4MPT
Tetrahydromethanopterin
-
CH
2=H4MPT N5,N10
Methylene tetrahydromethanopterin
-
CHH
4MPT+ N5,N10
Methenyltetrahydromethanopterin
-
MALDI-TOF-MS
Matrix-assisted laser desorption 相似文献
20.
Factor F430 is a yellow compound of unknown structure present in methanogenic bacteria. It has recently been shown to contain nickel. In this communication the influence of the nickel concentration in the growth medium on the factor F430 content of Methanobacterium thermoautotrophicum and on the nickel content of factor F430 was studied. It was found: (1) The content of factor F430 in the cells was strongly dependent on the nickel concentration of the growth medium. Cells grown on media with 2.5 M NiCl2 contained 28 times as much factor F430 per g as those grown on media with 0.075 M NiCl2; (2) factor F430 was synthesized in nickel deprived cells only upon the addition of nickel Nickel uptake paralleled factor F430 synthesis; (3) independent of the nickel concentration in the growth medium, the extinction coefficient at 430 nm of factor F430 per mol nickel was always near 22,500 cm-1 (mol Ni)-1. These findings indicate that nickel is an essential component of factor F430.Dedicated to Professor Otto Kandler on the occasion of his 60th birthday 相似文献