排序方式: 共有53条查询结果,搜索用时 15 毫秒
31.
S W Kengen P J Daas E F Duits J T Keltjens C van der Drift G D Vogels 《Biochimica et biophysica acta》1992,1118(3):249-260
Formaldehyde conversion into methyl-coenzyme M involves (a) reaction of the substrate with 5,6,7,8-tetrahydromethanopterin (H4MPT) giving 5,10-methylene-H4MPT, followed by its reduction to 5-methyl-H4MPT and (b) transfer of the methyl group from the latter compound to coenzyme M. The reactions were studied in a resolved system from Methanobacterium thermoautotrophicum strain delta H. The first part (a) of the reactions was catalyzed by the 55% ammonium sulfate supernatant of cell-free extracts. The methyltransferase step (b) was dependent on an oxygen-sensitive enzyme, called methyltransferase a (MTa). Isolation of MTa was achieved by gel filtration on Sephacryl S-400. MTa was a high-molecular-weight complex of at least 2000 kDa and between 900 to 1500 kDa when purified in the absence and presence of the detergent CHAPS, respectively. The enzyme consisted of 100 kDa units composed of three subunits in an alpha beta gamma configuration with apparent molecular masses of 35, 33 and 31 kDa, respectively. The corrinoid, 5-hydroxybenzymidazolyl cobamide (B12HBI, Factor III) copurified with MTa and the latter contained 2 nmol B12HBI per mg protein. B12HBI present in MTa could be methylated under the appropriate conditions by 5-methyl-H4MPT. These findings suggest that the corrinoid is a prosthetic group of MTa. MTa may be homologous to the corrinoid membrane protein purified before from M. thermoautotrophicum strain Marburg (Schulz, H., Albracht, S.P.J., Coremans, J.M.C.C. and Fuchs, G. (1988) Eur. J. Biochem. 171, 589-597). 相似文献
32.
Machielsen R Uria AR Kengen SW van der Oost J 《Applied and environmental microbiology》2006,72(1):233-238
The gene encoding a novel alcohol dehydrogenase that belongs to the aldo-keto reductase superfamily has been identified in the hyperthermophilic archaeon Pyrococcus furiosus. The gene, referred to as adhD, was functionally expressed in Escherichia coli and subsequently purified to homogeneity. The enzyme has a monomeric conformation with a molecular mass of 32 kDa. The catalytic activity of the enzyme increases up to 100 degrees C, and a half-life value of 130 min at this temperature indicates its high thermostability. AdhD exhibits a broad substrate specificity with, in general, a preference for the reduction of ketones (pH optimum, 6.1) and the oxidation of secondary alcohols (pH optimum, 8.8). Maximal specific activities were detected with 2,3-butanediol (108.3 U/mg) and diacetyl-acetoin (22.5 U/mg) in the oxidative and reductive reactions, respectively. Gas chromatrography analysis indicated that AdhD produced mainly (S)-2-pentanol (enantiomeric excess, 89%) when 2-pentanone was used as substrate. The physiological role of AdhD is discussed. 相似文献
33.
Hydrogenomics of the Extremely Thermophilic Bacterium Caldicellulosiruptor saccharolyticus
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Harmen J. G. van de Werken Marcel R. A. Verhaart Amy L. VanFossen Karin Willquist Derrick L. Lewis Jason D. Nichols Heleen P. Goorissen Emmanuel F. Mongodin Karen E. Nelson Ed W. J. van Niel Alfons J. M. Stams Donald E. Ward Willem M. de Vos John van der Oost Robert M. Kelly Serv W. M. Kengen 《Applied microbiology》2008,74(21):6720-6729
34.
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
35.
A bioinformatic screening of the genome of the hyperthermophilic bacterium Thermotoga maritima for ester-hydrolyzing enzymes revealed a protein with typical esterase motifs, though annotated as a hypothetical protein. To confirm its putative esterase function the gene (estD) was cloned, functionally expressed in Escherichia coli and purified to homogeneity. Recombinant EstD was found to exhibit significant esterase activity with a preference for short acyl chain esters (C4-C8). The monomeric enzyme has a molecular mass of 44.5 kDa and optimal activity around 95 degrees C and at pH 7. Its thermostability is relatively high with a half-life of 1 h at 100 degrees C, but less stable compared to some other hyperthermophilic esterases. A structural model was constructed with the carboxylesterase Est30 from Geobacillus stearothermophilus as a template. The model covered most of the C-terminal part of EstD. The structure showed an alpha/beta-hydrolase fold and indicated the presence of a typical catalytic triad consisting of a serine, aspartate and histidine, which was verified by site-directed mutagenesis and inhibition studies. Phylogenetic analysis showed that EstD is only distantly related to other esterases. A comparison of the active site pentapeptide motifs revealed that EstD should be grouped into a new family of esterases (Family 10). EstD is the first characterized member of this family. 相似文献
36.
Enrico Orsi Jules Beekwilder Dewi van Gelder Adèle van Houwelingen Gerrit Eggink Servé W.M. Kengen Ruud A. Weusthuis 《Microbial biotechnology》2020,13(4):1082-1093
Advances in synthetic biology and metabolic engineering have proven the potential of introducing metabolic by-passes within cell factories. These pathways can provide a more efficient alternative to endogenous counterparts due to their insensitivity to host's regulatory mechanisms. In this work, we replaced the endogenous essential 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in the industrially relevant bacterium Rhodobacter sphaeroides by an orthogonal metabolic route. The native 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway was successfully replaced by a heterologous mevalonate (MVA) pathway from a related bacterium. The functional replacement was confirmed by analysis of the reporter molecule amorpha-4,11-diene after cultivation with [4-13C]glucose. The engineered R. sphaeroides strain relying exclusively on the MVA pathway was completely functional in conditions for sesquiterpene production and, upon increased expression of the MVA enzymes, it reached even higher sesquiterpene yields than the control strain coexpressing both MEP and MVA modules. This work represents an example where substitution of an essential biochemical pathway by an alternative, heterologous pathway leads to enhanced biosynthetic performance. 相似文献
37.
Glycosaminoglycans (GAGs) are polysaccharides that are typically present in a wide diversity of animal tissue. Most common
GAGs are well-characterized and pharmaceutical applications exist for many of these compounds, e.g. heparin and hyaluronan. In addition, also bacterial glycosaminoglycan-like structures exist. Some of these bacterial GAGs
have been characterized, but until now no bacterial GAG has been found that possesses the modifications that are characteristic
for many of the animal GAGs such as sulfation and C5-epimerization. Nevertheless, the latter conversion may also occur in
bacterial and archaeal GAGs, as some prokaryotic polysaccharides have been demonstrated to contain L-iduronic acid. However,
experimental evidence for the enzymatic synthesis of L-iduronic acid in prokaryotes is as yet lacking. We therefore performed
an in silico screen for D-glucuronyl C5-epimerases in prokaryotes. Multiple candidate C5-epimerases were found, suggesting that many more
microorganisms are likely to exist possessing an L-iduronic acid residue as constituent of their cell wall polysaccharides. 相似文献
38.
Levisson M Han GW Deller MC Xu Q Biely P Hendriks S Ten Eyck LF Flensburg C Roversi P Miller MD McMullan D von Delft F Kreusch A Deacon AM van der Oost J Lesley SA Elsliger MA Kengen SW Wilson IA 《Proteins》2012,80(6):1545-1559
TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short‐chain acyl esters (C2–C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4‐nitrophenyl‐β‐D ‐xylopyranoside monoacetates as substrates in a β‐xylosidase‐coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine‐substituted and native structures of TM0077 were determined at 2.1 and 2.5 Å resolution, respectively, revealing a classic α/β‐hydrolase fold. TM0077 assembles into a doughnut‐shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 Å, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction. Proteins 2012. © 2012 Wiley Periodicals, Inc. 相似文献
39.
The hyperthermophilic archaeon Pyrococcus furiosus was found to form substantial amounts of l-alanine during batch growth on either cellobiose, maltose or pyruvate. Acetate, CO2 and H2 were produced next to alanine. The carbon- and electron balances were complete for all three substrates. Under standard growth conditions (N2/CO2 atmosphere) an alanine/acetate ratio of about 0.3 was found for either substrate. The alanine /acetate ratio was influenced, however, by the hydrogen partial pressure. In the presence of S0 or in coculture with Methanococcus jannaschii this ratio was only 0.07, whereas under a H2/CO2 atmosphere this ratio could amount up to 0.8. Alanine formation was also aflected by the NH
inf4
sup+
concentration, i.e. below 4 mM, NH
inf4
sup+
becomes limiting to alanine formation. Alanine formation was shown to occur via an alanine aminotransferase, which exhibited a specific activity in cell-free extract of up to 6.0 U/mg (90°C; direction of pyruvate formation). The alanine aminotransferase probably cooperates with glutamate dehydrogenase (up to 23 U/mg; 90°C) and ferredoxin: NADP+ oxidoreductase (up to 0.7 U/mg, using methyl viologen; 90°C) to recycle the electron acceptors involved in catabolism. Thus, the existence of this unusual alanine-forming branch enables P. furiosus to adjust its fermentation, depending on the redox potential of the terminal electron acceptor.Abbreviations DTT
dithiothreitol
- MV
methyl viologen
- AAT
alanine aminotransferase
- GDH
glutamate dehydrogenase
- MV: NADP+ OR
methyl viologen: NADP+ oxidoreductase 相似文献
40.
S W Kengen H W von den Hoff J T Keltjens C van der Drift G D Vogels 《BioFactors (Oxford, England)》1991,3(1):61-65
Factor F390 is the 8-OH adenylated form of the deazaflavin coenzyme F420, which is a central electron carrier in methanogenic bacteria. The enzymes catalysing the formation of F390 from ATP and F420 (F390 synthetase) and its hydrolysis into AMP and F420 (F390 hydrolase) were isolated and partially purified from Methanobacterium thermoautotrophicum. Both enzymes were oxygen-stable. The F390 synthetase tended to coelute with coenzyme F420 reducing hydrogenase during all purification steps. The 30-fold purified enzyme was still contaminated with the hydrogenase. The F390 hydrolase was purified 135-fold to a specific activity of 8.6 mumol/min/mg protein. The colourless enzyme consisted of one polypeptide of approximately 27,000 kd. 相似文献