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1.
Covalently linked fatty acids are increasingly recognized as an important type of post-translational protein modification. Many of such acylated proteins are found associated with cellular membranes. The membrane skeleton of the parasitic hemoflagellate Trypanosoma brucei consists of a regular array of microtubules which are tightly bound to the overlying cell membrane. A microtubule-binding protein (p41) has been identified within this structure which carries covalently bound fatty acid. The fatty acid linkage is sensitive to hydroxylamine treatment. After chemical transesterification, the released radioactivity co-migrates with fatty acid methyl esters in thin-layer chromatograms, establishing that the fatty acid was covalently bound to p41 via an ester (thioester) linkage. Upon detergent extraction of trypanosomes, p41 remains tightly bound to the cytoskeleton as long as Ca2+ ions are present. It can selectively be released from this structure by the addition of excess EGTA. Conversely, p41 binds to isolated cytoskeletons and to purified microtubules in vitro, the reaction again being entirely Ca2+-dependent.  相似文献   

2.
Menaquinol-fumarate oxidoreductase of Escherichia coli is a four-subunit membrane-bound complex that catalyzes the final step in anaerobic respiration when fumarate is the terminal electron acceptor. The catalytic domain of fumarate reductase consists of the FrdA subunit, which contains the active site, and a FAD prosthetic group covalently attached to His44, plus the FrdB subunit which contains at least two of the three nonidentical iron-sulfur clusters of the enzyme. To examine the role of covalently bound FAD in enzyme activity and electron transfer during anaerobic cell growth, site-directed mutagenesis was used to alter His44 of the FrdA subunit to a Ser, Cys, or Tyr residue. The resulting mutant enzyme complexes that were synthesized associated normally with the cytoplasmic membrane, but had decreased ability (greater than 70%) to reduce fumarate with reduced benzyl viologen, an artificial electron donor of low redox potential (Em = -359 mV; Clark, W. M. (1972) Oxidation-Reduction Potentials of Organic Systems, Robert E. Kreiger Publishing Co., Melbourne, FL). Even lower activities were measured when the higher potential, natural electron donor menaquinol was used, which, however, correlated with the slower growth rates of the different mutant complexes. In contrast to the normal enzyme, the mutant enzyme complexes were unable to oxidize succinate. Substitution of Arg for His44 produced a totally inactive enzyme complex that permitted no cell growth on nonfermentable substrates with fumarate as electron acceptor. All four mutant complexes contained noncovalently bound FAD in stoichiometric amounts. These data indicate a unique role of the 8 alpha-[N(3)-histidyl] FAD linkage in enzyme activity, by raising the redox potential of free FAD to permit reduction by both menaquinol and succinate.  相似文献   

3.
Vanillyl-alcohol oxidase was purified 32-fold from Penicillium simplicissimum, grown on veratryl alcohol as its sole source of carbon and energy. SDS/PAGE of the purified enzyme reveals a single fluorescent band of 65 kDa. Gel filtration and sedimentation-velocity experiments indicate that the purified enzyme exists in solution as an octamer, containing 1 molecule flavin/subunit. The covalently bound prosthetic group of the enzyme was identified as 8 alpha-(N3-histidyl)-FAD from pH-dependent fluorescence quenching (pKa = 4.85) and no decrease in fluorescence upon reduction with sodium borohydride. The enzyme shows a narrow substrate specificity, only vanillyl alcohol and 4-hydroxybenzyl alcohol are substrates for the enzyme. Cinnamyl alcohol is a strong competitive inhibitor of vanillyl-alcohol oxidation. The visible absorption spectrum of the oxidized enzyme shows maxima at 354 nm and 439 nm, and shoulders at 370, 417 and 461 nm. Under anaerobic conditions, the enzyme is easily reduced by vanillyl alcohol to the two-electron reduced form. Upon mixing with air, rapid reoxidation of the flavin occurs. Both with dithionite reduction and photoreduction in the presence of EDTA and 5-deazaflavin the red semiquinone flavin radical is transiently stabilized. Opposite to most flavoprotein oxidases, vanillyl-alcohol oxidase does not form a flavin N5-sulfite adduct. Photoreduction of the enzyme in the presence of the competitive inhibitor cinnamyl alcohol gives rise to a complete, irreversible bleaching of the flavin spectrum.  相似文献   

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5.
Rat brain mitochondria contain an NADH-linked nitro reductase that converts various aromatic nitro compounds, including the anti-schistosomal agent niridazole, into the hydroxylamine metabolites. The enzyme is tightly bound to the inner membrane and its activity is measurable only after disrupting the mitochondria. Triton X-100 (1%) and sonication partially solubilize the enzyme. The molecular weight determined by gel filtration is approx. 200 000. The temperature optima for the membrane-bound and for the solubilized enzyme are at 35 and 30 degrees C, respectively. The pH optimum for the membrane-bound enzyme is 9.2. NAD+ and 4-hydroxymercuribenzoate decrease the enzyme activity. Oxygen, carbon monoxide, cyanide, rotenone, barbiturates, chlorpromazine, dicumarol and chelating agents have no effect on the activity. The subcellular localization, substrate specificity and sensitivity to inhibitors distinguish the mitochondrial nitro reductase from the corresponding microsomal and cytosolic enzymes.  相似文献   

6.
The major UV-B photoproduct in DNA is the cyclobutane pyrimidine dimer (CPD). CPD-photolyases repair this DNA damage by a light-driven electron transfer. The chromophores of the class II CPD-photolyase from Arabidopsis thaliana, which was cloned recently [Taylor, R., Tobin, A. & Bray, C. (1996) Plant Physiol. 112, 862; Ahmad, M., Jarillo, J.A., Klimczak, L.J., Landry, L.G., Peng, T., Last, R.L. & Cashmore, A.R. (1997) Plant Cell 9, 199-207], have not been characterized so far. Here we report on the overexpression of the Arabidopsis CPD photolyase in Escherichia coli as a 6 x His-tag fusion protein, its purification and the analysis of the chromophore composition and enzymatic activity. Like class I photolyase, the Arabidopsis enzyme contains FAD but a second chromophore was not detectable. Despite the lack of a second chromophore the purified enzyme has photoreactivating activity.  相似文献   

7.
This paper reports the ligand binding properties of the major hemoglobin component from trout (Salmo irideus) covalently bound to a solid matrix (Sepharose or Sephadex). A comparison between the functional properties of this protein in solution and of the protein-matrix complex shows significant changes although the basic properties of the molecule are maintained on covalent binding to Sepharose (or Sephadex). Thus the Root effect, characteristic of Hb trout IV, is still present while the heme-heme interactions are, on the average, smaller in the matrix bound protein as compared to the soluble form. No differences in the O2 binding properties were observed when the protein was coupled to the resin, as the ligand bound or as the ligand free derivative. Although an unequivocal interpretation of the data is made difficult by the lack of information on the number and identity of the groups involved in the coupling, the main changes in the protein functional properties may be related to the chemical modifications "per se" more than to the immobilization imposed to the macromolecule by coupling to the matrix. Structural changes which mainly involve perturbation of the tertiary structure of the molecule may qualitatively rationalize the data.  相似文献   

8.
M C Bewley  C C Marohnic  M J Barber 《Biochemistry》2001,40(45):13574-13582
Cytochrome b5 reductase (cb5r) (EC 1.6.6.2) catalyzes the reduction of two molecules of cytochrome b5 using NADH as the physiological electron donor. The structure of pig cb5r at 2.4 A resolution was previously reported in the literature, but it was inconsistent with the biochemistry; for example, K83 and C245 were both implicated in the mechanism, but were not located at the active site. To address this problem, we have determined the structures of cb5r from rat at 2.0 A resolution and in a complex with NAD+ at 2.3 A resolution. We found significant differences throughout the rat structure compared to that of pig, including the locations of the lysine and cysteine residues mentioned above. To test the structural models, we made single amino acid substitutions of this lysine and showed that all substitutions produced correctly folded proteins and exhibited normal flavin behavior. However, the apparent kcat(NADH) decreased, and the apparent K(m) for NADH increased; the K(m)'s for cytochrome b5 were unchanged relative to that of the wild type. The largest effect was for the glutamate-substituted protein, which was further characterized using a charge transfer assay and found to be less efficient at NADH utilization than the wild type. These results are consistent with a role for this lysine in stabilizing the NADH-bound form of cb5r. We have concluded that the pig structure was mistraced in several regions and have reinterpreted mutants in these regions that give rise to the hereditary disease methemoglobinemia.  相似文献   

9.
delta-Aminolevulinic acid, the biosynthetic precursor of tetrapyrroles, is synthesized from glutamate via the tRNA-dependent five-carbon pathway in the green sulfur bacterium Chlorobium vibrioforme. The enzyme glutamyl-tRNA reductase (GTR), encoded by the hemA gene, catalyzes the first committed step in this pathway, which is the reduction of tRNA-bound glutamate to produce glutamate 1-semialdehyde. To characterize the GTR protein, the hemA gene from C. vibrioforme was cloned into expression plasmids that added an N-terminal His(6) tag to the expressed protein. The His-tagged GTR protein was purified using Ni affinity column chromatography. GTR was observable as a 49-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The native molecular mass, as determined by gel filtration chromatography, appeared to be approximately 40 kDa, indicating that native GTR is a monomer. However, when the protein was mixed with 5% (vol/vol) glycerol, the product had an apparent molecular mass of 95 kDa, indicating that the protein is a dimer under these conditions. Purified His(6)-GTR was catalytically active in vitro when it was incubated with Escherichia coli glutamyl-tRNA(Glu) and purified recombinant Chlamydomonas reinhardtii glutamate-1-semialdehyde aminotransferase. The expressed GTR contained 1 mol of tightly bound heme per mol of pep tide subunit. The heme remained bound to the protein throughout purification and was not removed by anion- or cation-exchange column chromatography. However, the bound heme was released during SDS-PAGE if the protein was denatured in the presence of beta-mercaptoethanol. Added heme did not inhibit the activity of purified expressed GTR in vitro. However, when the GTR was expressed in the presence of 3-amino-2,3- dihydrobenzoic acid (gabaculine), an inhibitor of heme synthesis, the purified GTR had 60 to 70% less bound heme than control GTR, and it was inhibited by hemin in vitro.  相似文献   

10.
YdhR is a 101-residue conserved protein from Escherichia coli. Sequence searches reveal that the protein has >50% identity to proteins found in a variety of other bacterial genomes. Using size exclusion chromatography and fluorescence spectroscopy, we determined that ydhR exists in a dimeric state with a dissociation constant of approximately 40 nM. The three-dimensional structure of dimeric ydhR was determined using NMR spectroscopy. A total of 3400 unambiguous NOEs, both manually and automatically assigned, were used for the structure calculation that was refined using an explicit hydration shell. A family of 20 structures was obtained with a backbone RMSD of 0.48 A for elements of secondary structure. The structure reveals a dimeric alpha,beta fold characteristic of the alpha+beta barrel superfamily of proteins. Bioinformatic approaches were used to show that ydhR likely belongs to a recently identified group of mono-oxygenase proteins that includes ActVA-Orf6 and YgiN and are involved in the oxygenation of polyaromatic ring compounds.  相似文献   

11.
12.
A multicomponent enzyme system that catalyzes the reduction of hydroxylamine and a number of its mono- and disubstituted derivatives by NADH has been isolated from microsomes. Three protein fractions isolated from pig liver microsomes are required to reconstitute NADH-hydroxylamine reductase activity. Two of the proteins appear identical with detergent-extracted cytochrome b5, and its flavoprotein reductase. The third protein fraction required for activity differs from previously isolated microsomal proteins. This fraction is free from detectable chromophores that absorb in the visible region of the spectrum and also appears free from metals. The properties of the NADH-hydroxylamine reductase reconstituted with the three components isolated from microsomes appears similar to the particle-bound system with respect to nucleotide and N-hydroxylamine substrate specificity.  相似文献   

13.
14.
15.
A specific mRNA for a structural lipoprotein of the Escherichiacoli outer membrane was translated in a wheat germ cell-free protein synthesizing system, S-adenosyl-methionine (SAM) and S-adenosyl-homocysteine (SAH) had neither stimulative nor inhibitory effect on the translation. When the products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two peaks appeared at the appearent molecular weights of about 15,000 and about 7,500. Both products were cross-reactive with antiserum against the lipoprotein.  相似文献   

16.
Thiamin dehydrogenase, a flavoprotein isolated from an unidentified soil bacterium, contains 1 mol of covalently bound FAD/mol of enzyme. A flavin peptide, isolated from tryptic-chymotryptic digests of the enzyme and hydrolyzed to the FMN level, shows a pH-dependent fluorescence yield being maximal at pH 3.5 to 4.0 and decreasing over 90% at pH 7.5 with a pKa of 5.8. Acid hydrolysis of the peptide results in an aminoacylflavin which shows a pKa of fluorescence quenching of 5.2. Absorption and electron paramagnetic resonance spectral data show the covalent substituent to be at the 8alpha position of the flavin as is the case with all known enzymes containing covalently bound flavin. The aminoacylflavin gives a negative Pauly reaction but yields 1 mol of histidine on drastic acid hydrolysis thus showing an imidazole ring nitrogen as the 8alpha substituent of the flavin. The aminoacylflavin differs from synthetic 8alpha-[N(3)-histidyl]riboflavin or its acid-modified form in pKa of fluorescence quenching, in electrophoretic mobility, in being reduced by borohydride, and in being labile to storage, yielding 8-formylriboflavin. In all of these properties, however, the 8alpha-histidylriboflavin isolated from thiamin dehydrogenase is indistinguishable from 8alpha-[N(1)-histidyl]riboflavin. It is therefore concluded that the FAD moiety of thiamin dehydrogenase is covalently linked via the 8alpha-methylene group to the N(1) position of the imidazole ring of histidine.  相似文献   

17.
Potassium tellurite (K2TeO3) is extremely toxic for most forms of life and only a limited number of organisms are naturally resistant to the toxic effects of this compound. Crude extracts prepared from the environmental isolate Aeromonas caviae ST catalize the in vitro reduction of in a NADH-dependent reaction. Upon fractionation by ionic exchange column chromatography three major polypeptides identified as the E1, E2, and E3 components of the pyruvate dehydrogenase (PDH) complex were identified in fractions exhibiting tellurite-reducing activity. Tellurite reductase and pyruvate dehydrogenase activities co-eluted from a Sephadex gel filtration column. To determine which component(s) of the PDH complex has tellurite reductase activity, the A. caviae ST structural genes encoding for E1 (aceE), E2 (aceF), and E3 (lpdA) were independently cloned and expressed in Escherichia coli and their gene products purified. Results indicated that tellurite reductase activity lies almost exclusively in the E3 component, dihydrolipoamide dehydrogenase. The E3 component of the PDH complex from E. coli, Zymomonas mobilis, Streptococcus pneumoniae, and Geobacillus stearothermophilus also showed NADH-dependent tellurite reductase in vitro suggesting that this enzymatic activity is widely distributed among microorganisms.  相似文献   

18.
A substantially improved purification of Escherichia coli NADH-dependent nitrite reductase was obtained by purifying it in presence of 1 mM-NO2- and 10 microM-FAD. The enzyme was obtained in 20% yield with a maximum specific activity of 1.04 kat . kg-1: more than 95% of this sample subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis migrated as a single band of protein. This highly active enzyme contained one non-covalently bound FAD molecule, and, probably, 5 Fe atoms and 4 acid-labile S atoms per subunit. No FMN, covalently bound flavin or Mo was detected. The spectrum of the enzyme shows absorption maxima at 386, 455, 530 and about 575 nm with a shoulder at 480--490 nm. The Soret-band/alpha-band absorbance ratio is about 4:1. These spectral features are characteristic of sirohaem, apart from the maximum at 455nm, which is attributed to flavin. The enzyme also catalyses the NADH-dependent reduction of horse heart cytochrome c, 2,6-dichlorophenol-indophenol and K3Fe(CN)6. The presence of sirohaem in E. coli nitrite reductase explains the apparent identity of the cysG and nirB gene of E. coli and inability of hemA mutants to reduce nitrite.  相似文献   

19.
The methylenetetrahydrofolate reductase from the carbon-monoxide-utilizing homoacetogen Peptostreptococcus productus (strain Marburg) has been purified to apparent homogeneity. The purified enzyme catalyzed the oxidation of NADH with methylenetetrahydrofolate as the electron acceptor at a specific activity of 380 mumols.min-1 mg protein-1 (37 degrees C; pH 5.5). The apparent Km for NADH was near 10 microM. The apparent molecular mass of the enzyme was determined by gel filtration to be approximately 250.0 kDa. The enzyme consists of eight identical subunits with a molecular mass of 32 kDa. It contains 4 FAD/mol octamer which were reduced by the enzyme with NADH as the electron donor; iron could not be detected. Oxygen had no effect on the enzyme. Ultracentrifugation of cell extracts revealed that about 40% of the enzyme activity was recovered in the particulate fraction, suggesting that the enzyme is associated with the membrane. The enzyme also catalyzed the methylenetetrahydrofolate reduction with methylene blue as an artificial electron donor. The oxidation of methyltetrahydrofolate was mediated with methylene blue as the electron acceptor; neither NAD+ nor viologen dyes could replace methylene blue in this reaction. NADP(H) or FAD(H2) were not used to substrates for the reaction in either direction. The activity of the purified enzyme, which was proposed to be involved in sodium translocation across the cytoplasmic membrane, was not affected by the absence or presence of added sodium. The properties of the enzyme differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase of the homoacetogen Clostridium formicoaceticum and of the NADP(+)-dependent reductase of eucaryotes investigated so far.  相似文献   

20.
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