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1.
Substitution of Azotobacter vinelandii hydrogenase small-subunit cysteines by serines can create insensitivity to inhibition by O2 and preferentially damages H2 oxidation over H2 evolution. 总被引:2,自引:1,他引:1
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Mutants in which conserved cysteines 294, 297 or 64 and 65 of the Azotobacter vinelandii hydrogenase small subunit were replaced by serines were studied. Cysteines 294 and 297 are homologous to cysteines 246 and 249 of the Desulfovibrio gigas hydrogenase, and these cysteines are ligands to the [3Fe-4S] clusters (A. Volbeda, M.-H. Charon, C. Piras, E. C. Hatchikian, M. Frey, and J. C. Fontecilla-Camps, Nature (London) 373:580-587, 1995). Cysteine 65 is homologous to cysteine 20 of the D. gigas hydrogenase, and this cysteine is a ligand to the proximal [4Fe-4S] cluster. All three mutants retained some hydrogenase activity. All three mutants studied had H2 oxidation-to-H2 evolution activity ratios with whole cells of approximately 1.5, compared with 46 for the wild type. The changes preferentially deplete H2 oxidation activity, while having less effect on evolution. The K64,65C-->S hydrogenase was partially purified and had a specific activity for the evolution reaction that was 22% that of the wild type, while the oxidation-specific activity was 2% that of the wild type. Because cysteine 65 provides a ligand to the proximal [4Fe-4S] cluster, this cluster can be altered without entirely eliminating enzyme activity. Likewise, the detection of H2 evolution and H2 oxidation activities with whole cells and membranes of the K294C-->S and K297C-->S mutants indicates that the [3Fe-4S] cluster can also be altered or possibly eliminated without entirely eliminating enzyme activity. Membranes with K294C-->S or K297C-->S hydrogenase were uninhibited by O2 in H2 oxidation and uninhibited by H2 in H2 evolution. Wild-type membranes and membranes with K64,65C-->S hydrogenase were both sensitive to these inhibitors. These data indicate that the [3Fe-4S] cluster controls the reversible inhibition of hydrogenase activity by O2 or H2. 相似文献
2.
Regulation of H2 oxidation activity and hydrogenase protein levels by H2, O2, and carbon substrates in Alcaligenes latus. 总被引:2,自引:3,他引:2
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Regulation of H2 oxidation activity and hydrogenase protein levels in the free-living hydrogen bacterium Alcaligenes latus was investigated. Hydrogenase activity was induced when heterotrophically grown cells were transferred to chemolithoautotrophic conditions, i.e., in the presence of H2 and absence of carbon sources, with NH4Cl as the N source. Under these conditions, H2 oxidation activity was detectable after 30 min of incubation and reached near-maximal levels by 12 h. The levels of hydrogenase protein, as measured by a Western blot (immunoblot) assay of the hydrogenase large subunit, increased in parallel with activity. This increase suggested that the increased H2 oxidation activity was due to de novo synthesis of hydrogenase protein. H2 oxidation activity was controlled over a surprisingly wide range of H2 concentrations, between 0.001 and 30% in the gas phase. H2 oxidation activity was induced to high levels between 2 and 12.5% O2, and above 12.5% O2, H2 oxidation activity was inhibited. Almost all organic carbon sources studied inhibited the expression of hydrogenase, although none repressed hydrogenase synthesis completely. In all cases examined, hydrogenase protein, as detected by Western blot, paralleled the level of H2 oxidation activity, suggesting that control of hydrogenase activity was mediated through changes in hydrogenase protein levels. 相似文献
3.
A M Brunner H Marquardt A R Malacko M N Lioubin A F Purchio 《The Journal of biological chemistry》1989,264(23):13660-13664
Three cysteine residues are located in the pro region of the transforming growth factor beta 1 (TGF-beta 1) precursor at amino acid positions 33, 223, and 225. Previous studies (Gentry, L. E., Lioubin, M. N., Purchio, A. F., and Marquardt, H. (1988) Mol. Cell. Biol. 8, 4162-4168) with purified recombinant TGF-beta 1 (rTGF-beta 1) precursor produced by Chinese hamster ovary (CHO) cells revealed that Cys-33 can form a disulfide bond with at least 1 cysteine residue in mature TGF-beta 1, contributing to the formation of a 90-110-kDa protein. We now show that Cys-223 and Cys-225 form interchain disulfide bonds. Site-directed mutagenesis was used to change these Cys codons to Ser codons, and mutant constructs were transfected into COS cells. Analysis of recombinant proteins by immunoblotting showed that by substituting Cys-33 the 90-110-kDa protein is not formed, and thus, more mature dimer (24 kDa) is obtained, corresponding to a 3- to 5-fold increase in biological activity. Substitution of Cys-223 and/or Cys-225 resulted in near wild-type levels of mature TGF-beta 1. Furthermore, cells transfected with plasmid coding for Ser at positions 223 and 225 expressed only monomeric precursor proteins and released bioactive TGF-beta 1 that did not require acid activation, suggesting that dimerization of the precursor pro region may be necessary for latency. 相似文献
4.
The role of amino acid residues in the H(2)-activating subunit (HoxH) of the NAD-reducing hydrogenase (SH) from Alcaligenes eutrophus has been investigated by site-directed mutagenesis. Conserved residues in the N-terminal L1 (RGxE) and L2 (RxCGxCx(3)H) and the C-terminal L5 (DPCx(2)Cx(2)H/R) motifs of the active site-harboring subunit were chosen as targets. Crystal structure analysis of the [NiFe] hydrogenase from Desulfovibrio gigas uncovered two pairs of cysteines (motifs L2 and L5) as coordinating ligands of Ni and Fe. Glutamate (L1) and histidine residues (L2 and L5) were proposed as being involved in proton transfer [Volbeda, A., Charon, M.-H., Piras, C., Hatchikian, E. C., Frey, M., and Fontecilla Camps, J. C. (1995) Nature 373, 580-587]. The A. eutrophus mutant proteins fell into three classes. (i) Replacement of the putative four metal-binding cysteines with serine led to the loss of H(2) reactivity and blocked the assembly of the holoenzyme. Exchange of Cys62, Cys65, or Cys458 was accompanied by the failure of the HoxH subunit to incorporate nickel, supporting the essential function of these residues in the formation of the active site. Although the fourth mutant of this class (HoxH[C461S]) exhibited nickel binding, the modified protein was catalytically inactive and unable to oligomerize. (ii) Mutations in residues possibly involved in proton transfer (HoxH[E43V], HoxH[H69L], and HoxH[H464L]) yielded Ni-containing proteins with residual low levels of hydrogenase activity. (iii) The most promising mutant protein (HoxH[R40L]), which was identified as a metal-containing tetrametric enzyme, was completely devoid of H(2)-dependent oxidoreductase activity but exhibited a remarkably high level of D(2)-H(+) exchange activity. These characteristics are compatible with the interpretation of a functional proton transfer uncoupled from the flow of electrons. 相似文献
5.
The oxygen-tolerant membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha H16 consists of three subunits. The large subunit HoxG carries the [NiFe] active site, and the small subunit HoxK contains three [FeS] clusters. Both subunits form the so-called hydrogenase module, which is oriented toward the periplasm. Membrane association is established by a membrane-integral cytochrome b subunit (HoxZ) that transfers the electrons from the hydrogenase module to the respiratory chain. So far, it was not possible to isolate the MBH in its native heterotrimeric state due to the loss of HoxZ during the process of protein solubilization. By using the very mild detergent digitonin, we were successful in isolating the MBH hydrogenase module in complex with the cytochrome b. H(2)-dependent reduction of the two HoxZ-stemming heme centers demonstrated that the hydrogenase module is productively connected to the cytochrome b. Further investigation provided evidence that the MBH exists in the membrane as a high molecular mass complex consisting of three heterotrimeric units. The lipids phosphatidylethanolamine and phosphatidylglycerol were identified to play a role in the interaction of the hydrogenase module with the cytochrome b subunit. 相似文献
6.
Characterization of the CO-induced, CO-tolerant hydrogenase from Rhodospirillum rubrum and the gene encoding the large subunit of the enzyme. 总被引:2,自引:2,他引:0
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In the presence of carbon monoxide, the photosynthetic bacterium Rhodospirillum rubrum induces expression of proteins which allow the organism to metabolize carbon monoxide in the net reaction CO + H2O --> CO2 + H2. These proteins include the enzymes carbon monoxide dehydrogenase (CODH) and a CO-tolerant hydrogenase. In this paper, we present the complete amino acid sequence for the large subunit of this hydrogenase and describe the properties of the crude enzyme in relation to other known hydrogenases. The amino acid sequence deduced from the CO-induced hydrogenase large-subunit gene (cooH) shows significant similarity to large subunits of other Ni-Fe hydrogenases. The closest similarity is with HycE (58% similarity and 37% identity) from Escherichia coli, which is the large subunit of an Ni-Fe hydrogenase (isoenzyme 3). The properties of the CO-induced hydrogenase are unique. It is exceptionally resistant to inhibition by carbon monoxide. It also exhibits a very high ratio of H2 evolution to H2 uptake activity compared with other known hydrogenases. The CO-induced hydrogenase is tightly membrane bound, and its inhibition by nonionic detergents is described. Finally, the presence of nickel in the hydrogenase is addressed. Analysis of wild-type R. rubrum grown on nickel-depleted medium indicates a requirement for nickel for hydrogenase activity. However, analysis of strain UR294 (cooC insertion mutant defective in nickel insertion into CODH) shows that independent nickel insertion mechanisms are utilized by hydrogenase and CODH. CooH lacks the C-terminal peptide that is found in other Ni-Fe hydrogenases; in other systems, this peptide is cleaved during Ni processing. 相似文献
7.
Functional analysis by site-directed mutagenesis of the NAD(+)-reducing hydrogenase from Ralstonia eutropha
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The tetrameric cytoplasmic [NiFe] hydrogenase (SH) of Ralstonia eutropha couples the oxidation of hydrogen to the reduction of NAD(+) under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site-directed mutagenesis in order to dissect the molecular mechanism of hydrogen activation. Based on phenotypic characterizations, 27 mutants were grouped into four different classes. Mutants of the major class, class I, failed to grow on hydrogen and were devoid of H(2)-oxidizing activity. In one of these isolates (HoxH I64A), H(2) binding was impaired. Class II mutants revealed a high D(2)/H(+) exchange rate relative to a low H(2)-oxidizing activity. A representative (HoxH H16L) displayed D(2)/H(+) exchange but had lost electron acceptor-reducing activity. Both activities were equally affected in class III mutants. Mutants forming class IV showed a particularly interesting phenotype. They displayed O(2)-sensitive growth on hydrogen due to an O(2)-sensitive SH protein. 相似文献
8.
The region including the conserved Ser65-Asp66 dipeptide in the tetracycline/H+ antiporter (TET) encoded by transposon Tn10 is thought to play a gating role (Yamaguchi, A., Ono, N., Akasaka, T., Noumi, T., and Sawai, T. (1990) J. Biol. Chem. 265, 15525-15530). The dipeptide is in putative interhelix loop2-3, which also includes the conserved sequence motif, GXXXXRXGRR, found in all TET proteins and sugar/H+ symporters. Through the combination of localized random and site-directed mutagenesis, each residue in loop2-3 was replaced. Among 10 residues in putative loop2-3, the important residues, of which substitution resulted in significant reduction or complete loss of the transport activity, were Gly62, Asp66, Gly69, and Arg70. The defect in the transport activity of the Gly62 and Gly69 substitution mutants corresponded to the steric hindrance by the substituents as to the putative beta-turn structure of the peptide backbone containing these glycines. Of 3 conserved Arg residues, the replacement of only Arg70 caused complete loss of the activity except for replacement with Lys, indicating the importance of a positive charge at this position, which is similar to the essentiality of a negative charge at Asp66. A "charge-neutralizing" intra-loop salt bridge between Asp66 and Arg70 was not likely because the double mutant in which Asp66 and Arg70 were replaced with asparagine and leucine, respectively, showed no transport activity. A triple mutant with only one positive charge at Arg70 in this loop showed about half the wild-type activity, indicating that the polycationic nature of the loop was not critical for the activity. Cys mutants as to the unessential residues in the loop were modifiable with N-ethylmaleimide, except for the Met64----Cys and Arg71----Cys mutants; however, the modification of only the Ser65----Cys mutant caused significant inhibition of the transport activity, indicating that position 65 is a unique position in the structure of loop2-3. 相似文献
9.
Role of cysteine residues in glutathione synthetase from Escherichia coli B. Chemical modification and oligonucleotide site-directed mutagenesis 总被引:2,自引:0,他引:2
H Kato T Tanaka T Nishioka A Kimura J Oda 《The Journal of biological chemistry》1988,263(24):11646-11651
Escherichia coli B glutathione synthetase is composed of four identical subunits; each subunit contains 4 cysteine residues (Cys-122, -195, -222, and -289). We constructed seven different mutant enzymes containing 3, 2, or no cysteine residues/subunit by replacement of cysteine codons with those of alanine in the gsh II gene using site-directed mutagenesis. Three mutant enzymes, Ala289, Ala222/289, Cys-free (Ala122/195/222/289), in which cysteine at residue 289 was replaced with alanine, were not inactivated by 5,5'-dithiobis(2-nitrobenzoate) (DTNB), while the other four mutants retaining Cys-289 were inactivated at the wild-type rate. From these selective inactivations of mutant enzymes by DTNB, the sulfhydryl group modified by DTNB was unambiguously identified as Cys-289. In this way, Cys-289 was found to be also a target of modification with 2-nitrothiocyanobenzoate and N-ethylmaleimide, while Cys-195 was of p-chloromercuribenzoate. These results suggest that both Cys-195 and Cys-289 were not essential for the activity of the glutathione synthetase, but chemical modification of either one of the two sulfhydryl groups resulted in complete loss of the activity. Replacement of Cys-122 to Ala-122 enhanced the reactivity of Cys-289 with sulfhydryl reagents. 相似文献
10.
Jörg Fischer Armin Quentmeier Susanne Kostka Regine Kraft C. G. Friedrich 《Archives of microbiology》1996,165(5):289-296
Hydrogenase of Thiobacillus ferrooxidans ATCC 19859 was purified from cells grown lithoautotrophically with 80% hydrogen, 8.6% carbon dioxide, and 11.4% air. Hydrogenase
was located in the 140,000 ×g supernatant in cell-free extracts. The enzyme was purified 7.3-fold after chromatography on Procion Red and Q-Sepharose with
a yield of 19%, resulting in an 85% pure preparation with a specific activity of 6.0 U (mg protein)–1. With native PAGE, a mol. mass of 100 and 200 kDa was determined. With SDS-PAGE, two subunits of 64 (HoxG) and of 34 kDa
(HoxK) were observed. Hydrogenase reacted with methylene blue and other artificial electron acceptors, but not with NAD. The
optimum of enzyme activity was at pH 9 and at 49° C. Hydrogenase contained 0.72 mol nickel and 6.02 mol iron per mol enzyme.
The relationship of the T. ferrooxidans hydrogenase to other proteins was examined. A 9.5-kb EcoRI fragment of T. ferrooxidans ATCC 19859 hybridized with a 2.2-kb XhoI fragment from Alcaligenes eutrophus encoding the membrane-bound hydrogenase. Antibodies against this enzyme did not react with the T. ferrooxidans hydrogenase in Western blot analysis. The N-terminal amino acid sequence (40 amino acids) of HoxK was 46% identical to that
of the hydrogen sensor HupU of Bradyrhizobium japonicum and 39% identical to that of the HupS subunit of the Desulfovibrio baculatus hydrogenase. The N-terminal sequence of 20 amino acids of HoxG of T. ferrooxidans was 83.3% identical to that of the 60-kDa subunit. HupL, of the hydrogenase of Anabaena sp. Sequences of ten internal peptides of HoxG were 50–100% identical to the respective sequences of HupL of the Anabaena sp. hydrogenase.
Received: 17 November 1995 / Accepted: 2 February 1996 相似文献
11.
Hydrogen-mediated enhancement of hydrogenase expression in Azotobacter vinelandii. 总被引:1,自引:1,他引:0
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Azotobacter vinelandii cultures express more H2 uptake hydrogenase activity when fixing N2 than when provided with fixed N. Hydrogen, a product of the nitrogenase reaction, is at least partly responsible for this increase. The addition of H2 to NH4+-grown wild-type cultures caused increased whole-cell H2 uptake activity, methylene blue-dependent H2 uptake activity of membranes, and accumulation of hydrogenase protein (large subunit as detected immunologically) in membranes. Both rifampin and chloramphenicol inhibited the H2-mediated enhancement of hydrogenase synthesis. Nif- A. vinelandii mutants with deletions or insertions in the nif genes responded to added H2 by increasing the amount of both whole-cell and membrane-bound hydrogenase activities. Nif- mutant strain CA11 contained fourfold more hydrogenase protein when incubated in N-free medium with H2 than when incubated in the same medium containing Ar. N2-fixing wild-type cultures that produce H2 did not increase hydrogenase protein levels in response to added H2. 相似文献
12.
Aerobically purified hydrogenase from Azotobacter vinelandii: activity, activation, and spectral properties 总被引:3,自引:0,他引:3
The hydrogenase from Azotobacter vinelandii is typically purified under anaerobic conditions. In this work, the hydrogenase was purified aerobically. The yields were low (about 2%) relative to those of the anaerobic purification (about 20%). The rate of enzyme activity depended upon the history of the enzyme. The enzyme preparations were active as isolated in H2 oxidation, and isotope exchange. The activity increased during the assay to a new maximal level (turnover activation). Treatment with reductants (e.g., H2, dithionite, dithiothreitol, indigo carmine) resulted in greater activation (reductant activation). Activation of the hydrogenase was accompanied by decrease in visible light absorption (300-600 nm) with maximal decreases at 450 and 345 nm which indicated the reduction of iron-sulfur clusters. The aerobically purified hydrogenase was susceptible to irreversible inactivation by cyanide. Pretreatment with acetylene did not influence activation of the hydrogenase. Once activated, the aerobically purified hydrogenase was indistinguishable from the anaerobically purified hydrogenase with respect to the catalytic properties tested. 相似文献
13.
Expression of uptake hydrogenase and hydrogen oxidation during heterotrophic growth of Bradyrhizobium japonicum. 总被引:8,自引:3,他引:5
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P van Berkum 《Journal of bacteriology》1987,169(10):4565-4569
Strains I-110 ARS, SR, USDA 136, USDA 137, and AK13 1c of Bradyrhizobium japonicum induced Hup activity when growing heterotrophically in medium with carbon substrate and NH4Cl in the presence of 2% H2 and 2% O2. Hup activity was induced during heterotrophic growth in the presence of carbon substrates, which were assimilated during the time of H2 oxidation. Strains I-110 ARS and SR grown heterotrophically or chemoautotrophically for 3 days had similar rates of H2 oxidation. Similar rates of Hup activity were also observed when cell suspensions were induced for 24 h in heterotrophic or chemoautotrophic growth medium with 1% O2, 10% H2, and 5% CO2 in N2. These results are contrary to the reported repression of Hup activity by carbon substrates in B. japonicum. Bradyrhizobial Hup activity during heterotrophic growth was limited by H2 and O2 and repressed by aerobic conditions, and CO2 addition had no effect. Nitrogenase and ribulosebisphosphate carboxylase activities were not detected in H2-oxidizing cultures of B. japonicum during heterotrophic growth. Immunoblot analysis of cell extracts with antibodies prepared against the 65-kilodalton subunit of uptake hydrogenase indicated that Hup protein synthesis was induced by H2 and repressed under aerobic conditions. 相似文献
14.
Aracena-Parks P Goonasekera SA Gilman CP Dirksen RT Hidalgo C Hamilton SL 《The Journal of biological chemistry》2006,281(52):40354-40368
The skeletal muscle Ca(2+)-release channel (ryanodine receptor type 1 (RyR1)) is a redox sensor, susceptible to reversible S-nitrosylation, S-glutathionylation, and disulfide oxidation. So far, Cys-3635 remains the only cysteine residue identified as functionally relevant to the redox sensing properties of the channel. We demonstrate that expression of the C3635A-RyR1 mutant in RyR1-null myotubes alters the sensitivity of the ryanodine receptor to activation by voltage, indicating that Cys-3635 is involved in voltage-gated excitation-contraction coupling. However, H(2)O(2) treatment of C3635A-RyR1 channels or wild-type RyR1, following their expression in human embryonic kidney cells, enhances [(3)H]ryanodine binding to the same extent, suggesting that cysteines other than Cys-3635 are responsible for the oxidative enhancement of channel activity. Using a combination of Western blotting and sulfhydryl-directed fluorescent labeling, we found that two large regions of RyR1 (amino acids 1-2401 and 3120-4475), previously shown to be involved in disulfide bond formation, are also major sites of both S-nitrosylation and S-glutathionylation. Using selective isotopecoded affinity tag labeling of RyR1 and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy, we identified, out of the 100 cysteines in each RyR1 subunit, 9 that are endogenously modified (Cys-36, Cys-315, Cys-811, Cys-906, Cys-1591, Cys-2326, Cys-2363, Cys-3193, and Cys-3635) and another 3 residues that were only modified with exogenous redox agents (Cys-253, Cys-1040, and Cys-1303). We also identified the types of redox modification each of these cysteines can undergo. In summary, we have identified a discrete subset of cysteines that are likely to be involved in the functional response of RyR1 to different redox modifications (S-nitrosylation, S-glutathionylation, and oxidation to disulfides). 相似文献
15.
Effect of nickel on activity and subunit composition of purified hydrogenase from Nocardia opaca 1 b 总被引:9,自引:0,他引:9
The NAD-reducing hydrogenase of Nocardia opaca 1 b was found to be a soluble, cytoplasmic enzyme. N. opaca 1 b does not contain an additional membrane-bound hydrogenase. The soluble enzyme was purified to homogeneity with a yield of 19% and a final specific activity of 45 mumol H2 oxidized min-1 mg protein-1. NAD reduction with H2 was completely dependent on the presence of divalent metal ions (Ni2+, Co2+, Mg2+, Mn2+) or of high salt concentrations (0.5-1.5 M). The most specific effect was caused by NiCl2, whose optimal concentration turned out to be 1 mM. The stimulation of activity by salts was the greater the less chaotrophic the anion. Maximal activity was achieved in 0.5 M potassium phosphate. Hydrogenase was also activated by protons. The pH optimum in 50 mM triethanolamine/HCl buffer containing 1 mM NiCl2 was 7.8-8.0. In the absence of Ni2+, hydrogenase was only active at pH values below 7.0. The reduction of other electron acceptors was not dependent on metal ions or salts, even though an approximately 1.5-fold stimulation of the reactions by 0.1-10 microM NiCl2 was observed. With the most effective electron acceptor, benzyl viologen, a 50-fold higher specific activity was determined than with NAD. The total molecular weight of hydrogenase has been estimated to be 200 000 (gel filtration) and 178 000 (sucrose density gradient centrifugation, and sodium dodecyl sulfate electrophoresis) respectively. The enzyme is a tetramer consisting of non-identical subunits with molecular weights of 64 000, 56 000, 31 000 and 27 000. It was demonstrated by electrophoretic analyses that in the absence of NiCl2 and at alkaline pH values the native hydrogenase dissociates into two subunit dimers. The first dimer was dark yellow coloured, completely inactive and composed of subunits with molecular weights of 64 000 and 31 000. The second dimer was light yellow, inactive with NAD but still active with methyl viologen. It was composed of subunits with molecular weights of 56 000 and 27 000. Immunological comparison of the hydrogenase of N. opaca 1 b and the soluble hydrogenase of Alcaligenes eutrophus H16 revealed that these two NAD-linked hydrogenases are partially identical proteins. 相似文献
16.
P Chiarugi R Marzocchini G Raugei C Pazzagli A Berti G Camici G Manao G Cappugi G Ramponi 《FEBS letters》1992,310(1):9-12
In this paper we describe the construction of five mutants of a bovine liver low M(r) phosphotyrosine protein phosphatase (PTPase) expressed as a fusion protein with the maltose binding protein in E. coli. Almost no changes in the kinetic parameters were observed in the fusion protein with respect to the native PTPase. Using oligonucleotide-directed mutagenesis Cys-17, Cys-62 and Cys-145 were converted to Ser while Cys-12 was converted to both Ser and Ala. The kinetic properties of the mutants, using p-nitrophenyl phosphate as substrate, were compared with those of the normal protein fused with the maltose binding protein of E. coli; both of the Cys-12 mutants showed a complete loss of enzymatic activity while the specific activity of the Cys-17 mutant was greatly decreased (200-fold). The Cys-62 mutant showed a 2.5-fold decrease in specific activity, while the Cys-145 mutant remained almost unchanged. These data confirm the involvement of Cys-12 and Cys-17 in the catalytic site and suggest that Cys-62 and Cys-145 mutations may destabilise the structure of the enzyme. 相似文献
17.
Periplasmic membrane-bound [NiFe]-hydrogenases undergo a complex maturation pathway, including cofactor incorporation, subunit assembly, and finally twin-arginine-dependent membrane translocation (Tat). In this study, the role of the two accessory proteins HoxO and HoxQ in the maturation of the membrane-bound [NiFe]-hydrogenase (MBH) of Ralstonia eutropha H16 was investigated. MBH activity was absent in soluble as well as membrane fractions of cells with deletions in the respective genes. The absence of HoxO and HoxQ led to degradation of the small subunit precursor (preHoxK) of the MBH. The two accessory proteins directly interacted with preHoxK prior to assembly of active MBH dimer in the cytoplasm. MBH mutants with modified Tat signal peptides were disrupted in preHoxK/HoxO/HoxQ complex formation. Isolated HoxO and HoxQ proteins formed a complex in vitro with the chemically synthesized HoxK Tat signal peptide. Two functions of the two chaperones are discussed: (i) protection of the Fe-S cluster containing HoxK subunit under oxygenic conditions, and (ii) avoidance of HoxK export prior to dimerization with the large MBH subunit HoxG. 相似文献
18.
Identification of nonessential disulfide bonds and altered conformations in the v-sis protein, a homolog of the B chain of platelet-derived growth factor. 总被引:7,自引:1,他引:6
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The protein encoded by v-sis, the oncogene of simian sarcoma virus, is homologous to the B chain of platelet-derived growth factor (PDGF). There are eight conserved Cys residues between PDGF-B and the v-sis protein. Both native PDGF and the v-sis protein occur as disulfide-bonded dimers, probably containing both intramolecular and intermolecular disulfide bonds. Oligonucleotide-directed mutagenesis was used to change the Cys codons to Ser codons in the v-sis gene. Four single mutants lacked detectable biological activity, indicating that Cys-127, Cys-160, Cys-171, and Cys-208 are required for formation of a biologically active v-sis protein. The other four single mutants retained biological activity as determined in transformation assays, indicating that Cys-154, Cys-163, Cys-164, and Cys-210 are dispensable for biological activity. Double and triple mutants containing three of these altered sites were constructed, some of which were transforming as well. The v-sis proteins encoded by biologically active mutants displayed significantly reduced levels of dimeric protein compared with the wild-type v-sis protein, which dimerized very efficiently. Furthermore, a mutant with a termination codon at residue 209 exhibited partial transforming activity. This study thus suggests that the minimal region required for transformation consists of residues 127 to 208. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the v-sis proteins encoded by some of the biologically active mutants exhibited an altered conformation when compared with the wild-type v-sis protein, and suggested that Cys-154 and Cys-163 participate in a nonessential disulfide bond. 相似文献
19.
Role of hydrogen in the activation and regulation of hydrogen oxidation by the soluble hydrogenase from Alcaligenes eutrophus H16. 总被引:1,自引:1,他引:0
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The activation kinetics of the H2-oxidizing activity of the soluble hydrogenase from Alcaligenes eutrophus H16 were investigated. Activation with Na2S2O4 plus 101 kPa H2 resulted in a rapid increase in activity over 1 h and constant activity after 3 h incubation. Less-stable activations were achieved if enzyme was incubated with Na2S2O4 under 1 kPa H2 or 101 kPa N2. The enzyme could also be partly activated either with NADH alone or with H2 alone. The level of activity obtained with both 101 kPa H2 and NADH present was greater than that obtained with either 101 kPa H2 or NADH alone. Activation with H2 plus NADH was virtually independent of NADH concentration but highly dependent on H2 concentration. The effects of various concentrations of H2 and constant concentration of NADH on the level of activation were the same whether H2 oxidation was assayed by H2-dependent Methylene Blue or NAD+ reduction. Diaphorase activity did not require activation and was little affected by the treatments that activated H2-oxidizing activity. The results suggest that H2 plays an important role in regulating the level of H2-oxidizing activity in this soluble hydrogenase. 相似文献
20.
Genetic and physiological characterization of the Rhodospirillum rubrum carbon monoxide dehydrogenase system. 总被引:1,自引:1,他引:0
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R L Kerby S S Hong S A Ensign L J Coppoc P W Ludden G P Roberts 《Journal of bacteriology》1992,174(16):5284-5294
A 3.7-kb DNA region encoding part of the Rhodospirillum rubrum CO oxidation (coo) system was identified by using oligonucleotide probes. Sequence analysis of the cloned region indicated four complete or partial open reading frames (ORFs) with acceptable codon usage. The complete ORFs, the 573-bp cooF and the 1,920-bp cooS, encode an Fe/S protein and the Ni-containing carbon monoxide dehydrogenase (CODH), respectively. The four 4-cysteine motifs encoded by cooF are typical of a class of proteins associated with other oxidoreductases, including formate dehydrogenase, nitrate reductase, dimethyl sulfoxide reductase, and hydrogenase activities. The R. rubrum CODH is 67% similar to the beta subunit of the Clostridium thermoaceticum CODH and 47% similar to the alpha subunit of the Methanothrix soehngenii CODH; an alignment of these three peptides shows relatively limited overall conservation. Kanamycin cassette insertions into cooF and cooS resulted in R. rubrum strains devoid of CO-dependent H2 production with little (cooF::kan) or no (cooS::kan) methyl viologen-linked CODH activity in vitro, but did not dramatically alter their photoheterotrophic growth on malate in the presence of CO. Upstream of cooF is a 567-bp partial ORF, designated cooH, that we ascribe to the CO-induced hydrogenase, based on sequence similarity with other hydrogenases and the elimination of CO-dependent H2 production upon introduction of a cassette into this region. From mutant characterizations, we posit that cooH and cooFS are not cotranscribed. The second partial ORF starts 67 bp downstream of cooS and would be capable of encoding 35 amino acids with an ATP-binding site motif. 相似文献