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1.
An NADP+ —dependent reversible 3-hydroxycarboxylate oxidoreductase present in Clostridium tyrobutyricum has been purified. As judged by gel electrophoresis the enzyme was pure after a 940-fold enrichment by four chromatographic steps. Its molecular mass was estimated to be 40–43 kDa. The enzyme was most active at pH 4.5 in the reduction of 3-oxobutyrate. Other substrates were 3-oxovalerate, 3-oxocaproate, 3-oxoisocaproate and 4-chloro-3-oxobutyrate. Except for the latter all substrates were converted enantioselectively to (S)-3-hydroxy acids in the presence of NADPH. 4-Chloro-3-oxobutyrate was reduced to the (R)-3-hydroxy acid. The specific activity of the enzyme was about 1400 mol min–1 mg–1 protein for the reduction of 3-oxobutyrate at pH 5.0. The Michaelis constant (K m) values for 3-oxobutyrate, 3-oxovalerate and 3-oxocaproate were determined to be 0.22, 1.6 and 3.0 mM, respectively. The K m values for dehydrogenation of (S)-3-hydroxybutyrate, (S)-3-hydroxyvalerate and (S)-3-hydroxycaproate were found to be 2.6, 1.1 and 5.2 mM, respectively. The identity of 43 of the first 45 N-terminal amino acid residues has been determined. So far such enzyme activities have been described in eucaryotes only.Dedicated to Prof. A. Trebst on the occasion of his 65th birthday  相似文献   

2.
A novel NADPH-dependent reductase (CaCR) from Candida albicans was cloned for the first time. It catalyzed asymmetric reduction to produce ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE). It contained an open reading frame of 843 bp encoding 281 amino acids. When co-expressed with a glucose dehydrogenase in Escherichia coli, recombinant CaCR exhibited an activity of 5.7 U/mg with ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. In the biocatalysis of COBE to (S)-CHBE, 1320 mM (S)-CHBE was obtained without extra NADP+/NADPH in a water/butyl acetate system, and the optical purity of the (S)-isomer was higher than 99% enantiomeric excess.  相似文献   

3.
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   

4.
The enzymatic production of (S)-4-bromo-3-hydroxybutyrate has been poorly studied compared with (S)-4-chloro-3-hydroxybutyrate. This can be attributed to the toxicity of bromide for biocatalysis. Recently, we isolated cDNA that encodes Penicillium citrinum β-keto ester reductase (KER) and the gene that encodes Leifsonia sp. alcohol dehydrogenase, which catalyzes the reduction of methyl 4-bromo-3-oxobutyrate to methyl (S)-4-bromo-3-hydroxybutyrate with high optical purity and productivity and expressed them in Escherichia coli. Moreover, protein engineering was performed using error-prone PCR-based random mutagenesis to improve the thermostability and enantioselectivity of KER. This review focuses on the establishment of a novel biotechnological process for the production of (S)-4-bromo-3-hydroxybutyrate using E. coli transformants. This process is suitable for industrial production of (S)-4-bromo-3-hydroxybutyrate, an intermediate for statin compounds.  相似文献   

5.
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   

6.
New triterpene glycosides, ulososides C, (20S,22S,23R,24S)-3,22,23-trihydroxy-3-O-(-D-glucopyranosyl)-32-nor-24-methyllanost-8(9)-ene-30-oic acid, D, (20S,22S,23R,24S)-3,22,23-trihydroxy-3-O-(-D-N-acetylglucosaminopyranosyl)-32-nor-24-methyllanost-8(9)-ene-30-oic acid, and E, (20S,22S,23R,24S)-3,22,23-trihydroxy-3-O-(-D-glucuronopyranosyl-(1 2)--D-arabinopyranosyl-32-nor-24-methyllanost-8(9)-ene-30-oic acid, were isolated from an Ulosa sp. sponge. Their structures were determined by spectral methods and chemical transformations. Specific features of their structures are discussed.  相似文献   

7.
A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis.  相似文献   

8.
The acetylation of 3-phenylthio-2-propanol (168 mg) was performed with vinyl acetate (1 ml) using different lipases from 15°C to 51°C. As a result, the (R)-enantiomer was selectively acetylated and the (S)-enantiomer was non-reactive in all the cases. An appropriate choice of conditions can be made to isolate both (R)-alcohol (ee 99%, 36 h, conversion 46%, sub/enz: 1/2) and (S)-alcohol (ee 93%, 38 h, conversion 46%, THF, sub/enz: 1 l–1) using Humicola lanuginosalipase (Lipolase). Increasing the amount of enzyme increased the ee.  相似文献   

9.
(+)-Isopiperitenone (100 mg l–1) was converted into (4S,6R)-6-hydroxy- and (4S,8R)-8,9-epoxyisopiperitenone, aside from the already known (+)-7-hydroxyisopiperitenone, by suspension cell culture of Mentha piperita. As (–)-isopiperitenone was hydroxylated similarly, this implies that the hydroxylating enzyme(s) have a broad substrate stereospecificity in regards to the stereochemistry at C4. (–)-(4R)-Carvone was reduced by the Mentha cells both at carbonyl and C1-C6 double bond to give (1R,2S,4R)-neodihydrocarveol and (1R,2R,4R)-dihydrocarveol with the former being the major product. (+)-(4S)-Carvone had a similar reduction pattern, producing (1S,2R,4S)-neodihydrocarveol and (1S,4S)-dihydrocarvone. Formation of these compounds indicates that the peppermint cell culture cannot only hydroxylate the allylic position but also reduce the ,-unsaturated carbonyl system.  相似文献   

10.
Summary Hemolymph was examined in young (ca. 86 days old), mature (ca. 163 days old), and old (ca. 294 days old) Aplysia for age-related changes in constituent proteins. In young, mature, and old animals protein concentrations were 1.6±0.27, 1.41±0.53, and 1.45±0.43 mg·ml-1, respectively. The copper-containing respiratory protein, hemocyanin, measured by determining the copper concentration, was found to increase significantly from young (0.98±0.51 g·ml-1) to mature (2.02±0.95 g·ml-1) Aplysia, with little change between mature and old (1.92±0.43 g·ml-1) animals. These findings were consistent with the results obtained when hemocyanin was directly measured by spectrophotometric absorption at 340 nm. Acetylcholinesterase (AChE) was present in the hemolymph of Aplysia. Its activity was highest in mature animals (3121±1627 units·mg-1) and least in old animals (1463±599 units·mg-1). Young animals had intermediate levels (2080±762 units·mg-1). SDS-PAGE revealed a distinct pattern of protein bands for hemolymph from each age group; hemolymph from the young group contained six prominent protein bands with molecular weights (MW) from 13 to 300 kDa. Hemolymph of mature and old animals exhibited four and three prominent protein bands, respectively, with MW between 45 and 300 kDa. A prominent band at 97 kDa was present in samples from the mature group, but was faint in samples from the old group and absent in samples from the young group. Under non-denaturing conditions the hemolymph protein band patterns for each group differed from the others, thereby demonstrating that the age-dependent differences in the protein profiles are intrinsic to hemolymph in vivo. Isoelectric focusing of the hemolymph samples revealed that the proteins were all acidic (pI ca. 3.0–6.5). The hemolymph from the young differed from the other two groups in having an additional acidic protein (pI ca. 4.0). A possible link between age-related changes in hemolymph proteins and age-related changes in the nervous system is proposed.Abbreviations 2-ME 2-mercaptoethanol - AChE acetylcholinesterase - FMRFamide amidated tetrapeptide containing phenylalanine, methionine, arginine and phenylalanine - MW molecular weight - PAS periodic acid-Schiff - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TRIS tris-(hydroxymethyl)-aminomethane  相似文献   

11.
A bacterium, strain 314B, able to assimilate (S)-5-oxo-2-tetrahydrofurancarboxylic acid was isolated from soil and identified as Erwinia cypripedii. A lactonase hydrolyzing (S)-5-oxo-2-tetrahydrofurancarboxylic acid to l--hydroxyglutaric acid was purified 63-fold with 2% recovery from crude extracts of this bacterium to homogeneity as judged by SDS-PAGE. The molecular masses estimated by SDS-PAGE and gel filtration were 41 kDa and 79 kDa, respectively. The maximum activity was observed at pH 6.5–7.5 and 35–45 °C. The enzyme showed lower activity toward dl-2-oxotetrahydrofuran-4,5-dicarboxylic acid, but did not act on (R)-5-oxo-2-tetrahydrofurancarboxylic acid and other natural and synthetic lactones tested.  相似文献   

12.
Cytidine-5-monophospho-N-acetylneuraminic acid:-galactoside 2-6sialyltransferase was purified from bovine colostrum by two sequential affinity chromatography steps on CDP-ethanolamine-Sepharose and CDP-ethanolamine-(N-caproylamino-)-Sepharose, respectively. While the conditions for elution were those of Paulsonet al. [J Biol Chem (1977) 252:3256–62], the ligand of the second affinity column was coupled to Sepharose by using 6-aminocaproic acid as linker. The ease of this procedure allows rapid synthesis of bulk quantities of ligand.Highly purified preparations of sialyltransferase were obtained which moved on gradient gel electrophoresis as a single band of 76 kDa and on dodecylsulphate electrophoresis as a single band of 54 kDa. The product of the reaction between lactose and CMP-N-acetylneuraminic acid catalyzed by the purified sialyltransferase was identified by high-resolution 500 MHz1H-NMR spectroscopy as Neu5Ac2-6Gal1-4Glc.  相似文献   

13.
A novel -keto ester reductase (KER) was purified to homogeneity from recombinant Escherichia coli (pTrcKER) cells, which efficiently expressed the ker gene cloned from Penicillium citrinum IFO4631. The enzyme was monomeric and had a molecular mass of 37 kDa. It catalyzed the reduction of some -keto esters, especially alkyl 4-halo-3-oxobutyrates. However, it did not catalyze the reverse reaction, the dehydrogenation of alkyl 4-halo-3-hydroxybutyrates and other alcohols. The enzyme required NADPH as a cofactor and showed no activity with NADH. Therefore, it was defined as a NADPH-dependent aldo–keto reductase (AKR3E1), belonging to the AKR superfamily. The enzyme stereospecifically produced methyl (S)-4-bromo-3-hydroxybutyrate from its keto derivative with high stereospecificity (97.9% enantiomer excess). E. coli cells expressing KER and glucose dehydrogenase in the water/butyl acetate two-phase system achieved a high productivity of (S)-4-bromo-3-hydroxybutyrate (277 mM, 54 mg/ml) in the organic solvent layer.  相似文献   

14.
Desulfovibrio vulgaris (Marburg) was grown on H2 plus sulfate and H2 plus thiosulfate as the sole energy sources and acetate plus CO2 as the sole carbon sources. Conditions are described under which the bacteria grew exponentially. Specific growth rates () and molar growth yields (Y) at different pH were determined. and Y were found to be strongly dependent on the pH. Highest growth rates and molar growth yields were observed for growth on H2 plus sulfate at pH 6.5 (=0.15h-1; Y SO 4 2- =8.3g·mol-1) and for growth on H2 plus thiosulfate at pH 6.8 (=0.21h-1; Y S 2O 3 2 =16.9g·mol-1).The growth yields were found to increase with increasing growth rates: plots of 1/Y versus 1/ were linear. Via extrapolation to infinite growth rates a Y SO4 2- /max of 12.2g·mol-1 and a YS2O 3 2- /max of 33.5g·mol-1 was obtained.The growth yield data are interpred to indicate that dissimilatory sulfate reduction to sulfide is associated with a net synthesis of 1 mol of ATP and that near to 3 mol of ATP are formed during dissimilatory sulfite reduction to sulfide.  相似文献   

15.
Anaerobic degradation of cresols by denitrifying bacteria   总被引:15,自引:0,他引:15  
The initial reactions in anaerobic metablism of methylphenols (cresols) and dimethylphenols were studied with denitrifying bacteria. A newly isolated strain, possibly a Paracoccus sp., was able to grow on o-or p-cresol as sole organic substrate with a generation time of 11 h; o-or p-cresol was completely oxidized to CO2 with nitrate being reduced to N2. A denitrifying Pseudomonas-like strain oxidized m-or p-cresol as the sole organic growth substrate completely to CO2 with a generation time of 14 h. Demonstration of intermediates and/or in vitro measurement of enzyme activities suggest the following enzymatic steps:(1) p-Cresol was metabolized by both strains via benzoyl-CoA as central intermediate as follows: p-cresol 4-OH-benzaldehyde 4-OH-benzoate 4-OH-benzoly-CoA benzoyl-CoA. Oxidation of the methyl group to 4-OH-benzaldehyde was catalyzed by p-cresol methylhydroxylase. After oxidation of the aldehyde to 4-OH-benzoate, 4-OH-benzoyl-CoA is formed by 4-OH-benzoyl-CoA synthetase; subsequent reductive dehydroxylation of 4-OH-benzoyl-CoA to benzoyl-CoA is catalyzed by 4-OH-benzoyl-CoA reductase (dehydroxylating).(2) o-Cresol was metabolized in the Paracoccus-like strain via 3-CH3-benzoyl-CoA as central intermediate as follows: o-cresol 4-OH-3-CH3-benzoate 4-OH-3-CH3-benzoyl-CoA 3-CH3-benzoyl-CoA. The following enzymes were demonstrated: (a) An enzyme catalyzing an isototope exchange reaction between 14CO2 and the carboxyl of 4-OH-3-CH3-benzoate; this activity is thought to be a partial reaction catalyzed by an o-cresol carboxylase. (b) 4-OH-3-CH3-benzoyl-CoA synthetase (AMP-forming) activating the carboxylation product 4-OH-3-CH3-benzoate to its coenzyme A thioester. (c) 4-OH-3-CH3-benzoyl-CoA reductase (dehydroxylating) catalyzing the reductive dehydroxylation of the 4-hydroxyl group with reduced benzyl viologen as electron donor to yield 3-CH3-benzoyl-CoA. This thioester may also be formed by action of a coenzyme A ligase when 3-CH3-benzoate is metabolized. 2,4-Dimethylphenol was metabolized via 4-OH-3-CH3-benzoate and further to 3-CH3-benzoyl-CoA.(3) The initial reactions of anaerobic metabolism of m-cresol in the Pseudomonas-like strain were not resolved. No indication for the oxidation of the methyl group nor for the carboxylation of m-cresol was found. In contrast, 2,4-and 3,4-dimethylphenol were oxidized to 4-OH-3-CH3-and 4-OH-2-CH3-benzoate, respectively, probably initiated by p-cresol methylhydroxylase; however, these compounds were not metabolized further.The hydroxyl and methyl groups are abbreviated as OH-and CH3-, respectively  相似文献   

16.
Hatanaka  Shin-Ichi  Furukawa  Jun  Aoki  Toshio  Akatsuka  Hirokazu  Nagasawa  Eiji 《Mycoscience》1994,35(4):391-394
Combining different chromatography systems, unusual nonprotein amino acids were isolated and unequivocally identified from a small amount (less than 100 g fresh weight) ofAmanita gymnopus fruit body. Without obtaining crystals of these amino acids, on the basis of1H-NMR determination, high resolution mass spectrometry, chlorine analysis and oxidation with L-amino acid oxidase, one of them proved to be a new chloroamino acid, (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid (G2). The other three were (2S)-2-amino-5-hexenoic acid (G1), (2S)-2-amino-4,5-hexadienoic acid (G3) and (2S)-2-amino-5-hexynoic acid (G4). Amino acid (G1) was also encountered for the first time in natural products. Amino acid (G3) has been reported from several kinds of fungi belonging toAmanita, subgenusLepidella. The occurrence of amino acid (G4) was already reported fromCortinarius claricolor.Part 23 in the series Biochemical studies of nitrogen compounds in fungi. Part 22, Hatanaka, S. I. et al. 1985. Trans. Mycol. Soc. Japan26: 61–68.  相似文献   

17.
Membrane preparations of Fusobacterium nucleatum grown on glutamate contain glutaconyl-CoA decarboxylase at a high specific activity (13.8 nkat/mg protein). The enzyme was solubilized with 2% Triton X-100 in 0.5M NaCl and purified 63-fold to a specific activity of 870 nkat/mg by affinity chromatography on monomeric avidin-Sepharose. The activity of the decarboxylase was strictly dependent on Na+ (K m=3 mM) and was stimulated up to 3-fold by phospholipids. The glutaconyl-CoA decarboxylases from the gram-positive bacteria Acidaminococcus fermentans and Clostridium symbiosum have a lower apparent K m for Na+ (1 mM) and were not stimulated by phospholipids. In addition only the fusobacterial decarboxylase required sodium ion for stability and was inactivated by potassium ion. By incorporation of this purified enzyme into phospholipids an electrogenic sodium ion pump was reconstituted. The enzyme consists of four subunits, (m=65 kDa), (33 kDa), (19 kDa), and (16 kDa) with the functions of a carboxy transferase (), a carboxy lyase ( and probably ) and a biotin carrier (). The subunits are very similar to those of the glutaconyl-CoA decarboxylases from the gram-positive bacteria. With an antiserum directed against the decarboxylase from A. fermentans the - and the biotin containing subunits of the three decarboxylases and that from Peptostreptoccus asaccharolyticus could be detected on Western blots.  相似文献   

18.
An optimized repeated-fed-batch fermentation process for the synthesis of dihydroxyacetone (DHA) from glycerol utilizing Gluconobacter oxydans is presented. Cleaning, sterilization, and inoculation procedures could be reduced significantly compared to the conventional fed-batch process. A stringent requirement was that the product concentration was kept below a critical threshold level at all times in order to avoid irreversible product inhibition of the cells. On the basis of experimentally validated model calculations, a threshold value of about 60 kg m-3 DHA was obtained. The innovative bioreactor system consisted of a stirred tank reactor combined with a packed trickle-bed column. In the packed column, active cells could be retained by in situ immobilization on a hydrophilized Ralu-ring carrier material. Within 17 days, the productivity of the process could be increased by 75% to about 2.8 kg m-3 h-1. However, it was observed that the maximum achievable productivity had not been reached yet.Abbreviations K O Monod half saturation constant of dissolved oxygen (kg m-3) - K S Monod half saturation constant of substrate glycerol (kg m-3) - O Dissolved oxygen concentration (kg m-3) - P Product concentration (kg m-3) - P crit Critical product concentration constant (kg m-3) - S Substrate concentration (kg m-3) - t Time (s) - X Biomass concentration (dry weight) (kg m-3) - Y P/S Yield coefficient of product from substrate - Y X/S Yield coefficient of biomass from substrate - Growth dependent specific production rate constant (kg m-3) - Growth independent specific production rate constant (s-1) - Specific growth rate (s-1) - max Maximum specific growth rate constant (s-1)  相似文献   

19.
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   

20.
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   

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