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IscU as a scaffold for iron-sulfur cluster biosynthesis: sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU 总被引:12,自引:0,他引:12
Iron-sulfur cluster biosynthesis in both prokaryotic and eukaryotic cells is known to be mediated by two highly conserved proteins, termed IscS and IscU in prokaryotes. The homodimeric IscS protein has been shown to be a cysteine desulfurase that catalyzes the reductive conversion of cysteine to alanine and sulfide. In this work, the time course of IscS-mediated Fe-S cluster assembly in IscU was monitored via anaerobic anion exchange chromatography. The nature and properties of the clusters assembled in discrete fractions were assessed via analytical studies together with absorption, resonance Raman, and M?ssbauer investigations. The results show sequential cluster assembly with the initial IscU product containing one [2Fe-2S](2+) cluster per dimer converting first to a form containing two [2Fe-2S](2+) clusters per dimer and finally to a form that contains one [4Fe-4S](2+) cluster per dimer. Both the [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU are reductively labile and are degraded within minutes upon being exposed to air. On the basis of sequence considerations and spectroscopic studies, the [2Fe-2S](2+) clusters in IscU are shown to have incomplete cysteinyl ligation. In addition, the resonance Raman spectrum of the [4Fe-4S](2+) cluster in IscU is best interpreted in terms of noncysteinyl ligation at a unique Fe site. The ability to assemble both [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU supports the proposal that this ubiquitous protein provides a scaffold for IscS-mediated assembly of clusters that are subsequently used for maturation of apo Fe-S proteins. 相似文献
3.
Jensen KP 《Journal of inorganic biochemistry》2008,102(1):87-100
This work reports density functional computations of metal-substituted models of biological [Fe3S4] clusters in oxidation states [MFe2S4](+/0/-1) (M=Mn, Fe, Co, Ni, Cu, Zn, and Mo). Geometry optimization with a dielectric screening model is shown to provide a substantial improvement in structure, compared to earlier used standard procedures. The error for average Fe-S bonds decreased from 0.038A to 0.016A with this procedure. Four density functionals were compared, B3LYP, BP86, TPSS, and TPSSh. B3LYP and to a lesser extent TPSSh energies were inconsistent with experiment for the oxidized [Fe3S4]+ cluster. BP86 (and to a slightly lesser extent TPSS) was within expected theoretical and experimental uncertainties for all oxidation states, the only qualitative error being 5kJ/mol in favor of the M(S)=3/2 configuration for the [Fe3S4]+ cluster, so BP86 was used for quantitative results. Computed reorganization energies and reduction potentials point directly towards the [Fe3S4] cluster as the superior choice of electron carrier, with the [ZnFe2S4] cluster a close second. In addition, partially and fully Mo-substituted clusters were investigated and found to have very low reorganization energies but too negative reduction potentials. The results provide a direct rationale why any substitution weakens the cluster as an electron carrier, and thus why the [Fe3S4] composition is optimal in the biological clusters. 相似文献
4.
Muro F Iimura S Yoneda Y Chiba J Watanabe T Setoguchi M Iigou Y Takayama G Yokoyama M Takashi T Nakayama A Machinaga N 《Bioorganic & medicinal chemistry》2008,16(23):9991-10000
Optimization of benzoic acid derivatives by introducing substituents into the diphenyl urea moiety led to the identification of compound 20l as a potent VLA-4 antagonist. Compound 20l inhibited eosinophil infiltration into bronchial alveolar lavage fluid in a murine asthma model by oral dosing and its efficacy was comparable to anti-mouse alpha4 antibody (R1-2). Furthermore, this compound significantly blocked bronchial hyper-responsiveness in the model. 相似文献
5.
James A. Fee Jesus M. Castagnetto David A. Case Louis Noodleman C. David Stout Rhonda A. Torres 《Journal of biological inorganic chemistry》2003,8(5):519-526
The geometry proposition that "four points not in a plane describe one and only one sphere" provides a novel tool for analyzing protein-induced distortions in [4Fe-4S] clusters. A geometrically perfect reference structure comprises interlaced, regular tetrahedra of Fe, S, and S gamma atoms having T(d) symmetry. Three circumspheres are defined by the three sets of four atoms, the circumcenters of which are unique points within the cluster. The structure is thus re-defined by the positions of the circumcenters in xyz space and the r, theta, phi of each atom on its respective sphere. Analysis of 12 high-resolution structures of protein-bound and small molecule [4Fe-4S](SR)(4) clusters revealed: (a) the circumcenters are generally non-coincident by approximately 0.01 to approximately 0.06 A; (b) the Fe radius, r(Fe), is nominally independent of core oxidation state, having values between 1.66 to 1.69 A, whereas r(S) and r(SG), which have ranges of 2.18-2.24 A and 3.87-3.94 A, respectively, both increase by as much as approximately 3% upon reduction from the 3+ to the 1+ core valence; (c) deviation of some atoms from the theta, phi of a perfect tetrahedron can be large, approximately 10 degrees, and sets of atoms can show patterns of motion on their spheres that result from changes in Fe-S bond lengths. Density functional theory calculations suggest that the [4Fe-4S] core itself requires rather little energy to distort (approximately 2 kcal/mol), whereas significantly more energy is required to distort the Sgamma shell (~4 kcal/mol) to that of cluster I in Clostridium acidurici ferredoxin. 相似文献
6.
The ability of myoglobin (Mb) to reversibly bind O2 and other ligands has been well characterized. Mb also participates with a variety of redox metals to form metmyoglobin (metMb). By using an anaerobic stopped-flow device we have measured outer-sphere oxidation by [Fe(CN)6]3 of native sperm whale myoglobin, recombinant wild-type Mb, and a series of mutant Mb proteins in which the distal His-64 was changed to Gly, Phe, Leu or Val. Second-order rate constants for oxidation of mutant proteins are 10-15 times greater than for recombinant or native (kox approximately 10(6) M-1 s-1). We attribute the reduced rate of oxidation of wild-type protein to a higher reorganization energy imposed by the presence of the unique water/His-64/heme interaction, which is absent in the mutant proteins. 相似文献
7.
In this study we report the in vitro inhibition of leukotriene synthesis in calcium ionophore (A23187)-stimulated, intact human blood neutrophils by AHR-5333. The results showed that AHR-5333 inhibits 5-HETE, LTB4 and LTC4 synthesis with IC50 values of 13.9, 13.7 and 6.9 microM, respectively. Further examination of the effect of AHR-5333 on individual reactions of the 5-lipoxygenase pathway (i.e. conversion of LTA4 to LTB4, LTA4 to LTC4, and arachidonic acid to 5-HETE) showed that this agent was not inhibitory to LTA4 epoxyhydrolase and glutathione-S-transferase activity in neutrophil homogenates. However, conversion of arachidonic acid (30 microM) to 5-HETE was half maximally inhibited by 20 microM AHR-5333 in the cell-free system. The inhibition of LTB4 and LTC4 formation in intact neutrophils by AHR-5333 appears to be entirely due to a selective inhibition of 5-lipoxygenase activity and an impaired formation of LTA4, which serves as substrate for LTA4 epoxyhydrolase and glutathione-S-transferase. AHR-5333 did not affect the transformation of exogenous arachidonic acid to thromboxane B2, HHT and 12-HETE in preparations of washed human platelets, indicating that this agent has no effect on platelet prostaglandin H synthase, thromboxane synthase and 12-lipoxygenase activity. The lack of inhibitory activity of AHR-5333 on prostaglandin H synthase activity was confirmed with microsomal preparations of sheep vesicular glands. 相似文献
8.
Jin Z Heinnickel M Krebs C Shen G Golbeck JH Bryant DA 《The Journal of biological chemistry》2008,283(42):28426-28435
The NfuA protein has been postulated to act as a scaffolding protein in the biogenesis of photosystem (PS) I and other iron-sulfur (Fe/S) proteins in cyanobacteria and chloroplasts. To determine the properties of NfuA, recombinant NfuA from Synechococcus sp. PCC 7002 was overproduced and purified. In vitro reconstituted NfuA contained oxygen- and EDTA-labile Fe/S cluster(s), which had EPR properties consistent with [4Fe-4S] clusters. After reconstitution with 57Fe2+, M?ssbauer studies of NfuA showed a broad quadrupole doublet that confirmed the presence of [4Fe-4S]2+ clusters. Native gel electrophoresis under anoxic conditions and chemical cross-linking showed that holo-NfuA forms dimers and tetramers harboring Fe/S cluster(s). Combined with iron and sulfide analyses, the results indicated that one [4Fe-4S] cluster was bound per NfuA dimer. Fe/S cluster transfer from holo-NfuA to apo-PsaC of PS I was studied by reconstitution of PS I complexes using P700-F(X) core complexes, PsaD, apo-PsaC, and holo-NfuA. Electron transfer measurements by time-resolved optical spectroscopy showed that holo-NfuA rapidly and efficiently transferred [4Fe-4S] clusters to PsaC in a reaction that required contact between the two proteins. The NfuA-reconstituted PS I complexes had typical charge recombination kinetics from [F(A)/F(B)](-) to P700+ and light-induced low-temperature EPR spectra. These results establish that cyanobacterial NfuA can act as a scaffolding protein for the insertion of [4Fe-4S] clusters into PsaC of PS I in vitro. 相似文献
9.
Setoguchi M Iimura S Sugimoto Y Yoneda Y Chiba J Watanabe T Muro F Iigo Y Takayama G Yokoyama M Taira T Aonuma M Takashi T Nakayama A Machinaga N 《Bioorganic & medicinal chemistry》2012,20(3):1201-1212
For the purpose of obtaining orally potent VLA-4 inhibitors, we have carried out structural modification of the (N'-phenylureido)phenyl group in compound 1, where the group was found to be attributed to poor pharmacokinetic profile in our previous research. Through modification, we have identified several compounds with both potent in vitro activity and improved oral exposure. In particular, compound 7e with 7-fluoro-2-(1-methyl-1H-indol-3-yl)-1,3-benzoxazolyl group as a novel replacement of the (N'-phenylureido)phenyl group significantly inhibited eosinophil infiltration into bronchoalveolar lavage fluid at 15mg/kg in an Ascaris-antigen-induced murine bronchial inflammatory model, and its efficacy was comparable to that of the anti-mouse α(4) antibody (R1-2). 相似文献
10.
Chern JH Chang CS Tai CL Lee YC Lee CC Kang IJ Lee CY Shih SR 《Bioorganic & medicinal chemistry letters》2005,15(19):4206-4211
The new pyridyl imidazolidinone derivative, 1-[5-(4'-chlorobiphenyl-4-yloxy)-3-methylpentyl]-3-pyridin-4-yl-imidazolidin-2-one (+/-)-1a, was synthesized and found to have an excellent antiviral activity against EV71 (IC50 = 0.009 microM). Therefore, both the enantiomers, (S)-(+)-1a and (R)-(-)-1a, have been prepared starting from readily available monomethyl (R)-3-methylglutarate (7) as a useful chiral building block and their antiviral activity was evaluated in a plaque reduction assay. Interestingly, we observed that the enantiomer (S)-(+)-1a was 10-fold more active against enterovirus71 (EV71) (IC50 = 0.003 microM) than the corresponding enantiomer (R)-(-)-1a (IC50 = 0.033 microM). Similar results were found against all five strains (1743, 2086, 2231, 4643, and BrCr) of EV71 tested. This demonstrated that the absolute configuration of the chiral carbon atom at the 3-position of the alkyl linker considerably influenced the anti-EV71 activity of these pyridyl imidazolidinones. 相似文献
11.
It is shown that the previously characterized [Fe(III)(SR)(4)](1-) (R=Et, i-Pr, Ph) complexes can be synthesized by the direct reaction of 4equiv. of LiSR with FeCl(3) in DMF solution. [Fe(III)(SR)(4)](1-) complexes are synthetic analogs for the [Fe(III)(S-Cys)(4)] center in rubredoxin proteins. 相似文献
12.
Pan W Miao HQ Xu YJ Navarro EC Tonra JR Corcoran E Lahiji A Kussie P Kiselyov AS Wong WC Liu H 《Bioorganic & medicinal chemistry letters》2006,16(2):409-412
A novel class of 1-[4-(1H-benzoimidazol-2-yl)-phenyl]-3-[4-(1H-benzoimidazol-2-yl)-phenyl]-ureas are described as potent inhibitors of heparanase. Among them are 1,3-bis-[4-(1H-benzoimidazol-2-yl)-phenyl]-urea (7a) and 1,3-bis-[4-(5,6-dimethyl-1H-benzoimidazol-2-yl)-phenyl]-urea (7d), which displayed good heparanase inhibitory activity (IC(50) 0.075-0.27 microM). Compound 7a showed good efficacy in a B16 metastasis model. 相似文献
13.
《BBA》1987,891(1):94-98
Core extrusion of the bound iron-sulfur centers from spinach Photosystem I showed the presence of [2Fe-2S] clusters as well as [4Fe-4S] clusters among FA, FB and FX. Extrusion of the iron-sulfur ensemble was not quantitative; however, the presence of [2Fe-2S] clusters correlated with higher concentration of unfolding solvent. Since FX is highly resistant to denaturation, and since FA and FB are known to contain [4Fe-4S] clusters, the [2Fe-2S] clusters are assigned to FX. The presence of [2Fe-2S] clusters in Photosystem I has significance in the structure and organization of FX on the reaction center. Since four cysteinyl ligands are assumed to hold an iron-sulfur cluster, a Photosystem I subunit may consist of two approx. 64-kDa proteins bridged by a single [2Fe-2S] cluster. The complete reaction center would consist of two subunits positioned so that two [2Fe-2S] clusters are in magnetic interaction, thereby constituting FX. 相似文献
14.
Matulenko MA Surber B Fan L Kolasa T Nakane M Terranova MA Uchic ME Miller LN Chang R Donnelly-Roberts DL Namovic MT Moreland RB Brioni JD Stewart AO 《Bioorganic & medicinal chemistry letters》2004,14(20):5095-5098
The first selective dopamine D4 agonist radioligand is described. The synthesis of these piperazine radioligands relied on the transformation of brominated precursors 4a and 4b with tritium gas in the presence of a sensitive cyano functional group. The specific activity of these two radioligands was measured and [3H]6b found to be suitable for use in D4 saturation and competition binding studies. The synthesis, biological, and radioactivity of this new agonist radioligand as well as preliminary SAR will be discussed. 相似文献
15.
《Journal of Molecular Catalysis .B, Enzymatic》2011,68(3-4):266-270
The asymmetric microbial reduction of 1-(4-fluorophenyl)-3(R)-[3-oxo-3-(4-fluorophenyl)-propyl]-4(S)-(4-hydroxyphenyl)azetidin-2-one to 1-(4-fluorophenyl)-3(R)-[3(S)-hydroxy-3-(4-fluorophenyl)-propyl]-4(S)-(4-hydroxyphenyl)azetidin-2-one (Ezetimibe) by Rhodococcus fascians MO22 is described. The catalytic capability of the microorganism for reduction has been examined also with protected ketone, an intermediate from chemical synthesis of Ezetimibe. Various parameters of the bioreduction have been optimized: the strain converted 94.8% of ketone and 63% of protected ketone into Ezetimibe with the same de of 99.9%. In the later case, two chemical steps are replaced with a single biotransformation. 相似文献
16.
Markus Wollenberg Carsten Berndt Eckhard Bill Jens D Schwenn Andreas Seidler 《European journal of biochemistry》2003,270(8):1662-1671
Two proteins with similarity to IscA are encoded in the genome of the cyanobacterium Synechocystis PCC 6803. One of them, the product of slr1417 which accounts for 0.025% of the total soluble protein of Synechocystis was over-expressed in E. coli and purified. The purified protein was found to be mainly dimeric and did not contain any cofactor. Incubation with iron ions, cysteine and Synechocystis IscS led to the formation of one [2Fe2S] cluster at an IscA dimer as demonstrated (by the binding of about one iron and one sulfide ion per IscA monomer) by UV/Vis, EPR and M?ssbauer spectroscopy. M?ssbauer spectroscopy further indicated that the FeS cluster was bound by four cysteine residues. Site-directed mutagenesis revealed that of the five cysteine residues only C110 and C112 were involved in cluster binding. It was therefore concluded that the [2Fe2S] cluster is located between the two protomers of the IscA dimer and ligated by C110 and C112 of both protomers. The cluster could be transferred to apo ferredoxin, a [2Fe2S] protein, with a half-time of 10 min. Surprisingly, incubation of cluster-containing IscA with apo adenosine 5'-phosphosulfate reductase led to a reactivation of the enzyme which requires the presence of a [4Fe4S] cluster. This demonstrates that it is possible to build [4Fe4S] clusters from [2Fe2S] units. 相似文献
17.
Barbakadze V. V. Kemertelidze E. P. Targamadze I. L. Shashkov A. S. Usov A. I. 《Russian Journal of Bioorganic Chemistry》2002,28(4):326-330
Two high-molecular water-soluble preparations with high anticomplementary and antioxidant activity were isolated from the roots of Symphytum asperum and S. caucasicum. Their main chemical constituent was found to be poly[oxy-1-carboxy-2-(3,4-dihydroxyphenyl)ethylene] according to IR and NMR spectroscopy. 相似文献
18.
Sandro Boland Olivier Defert Jo Alen Arnaud Bourin Karolien Castermans Nele Kindt Nicki Boumans Laura Panitti Sarah Van de Velde Ingeborg Stalmans Dirk Leysen 《Bioorganic & medicinal chemistry letters》2013,23(23):6442-6446
Clinical development of ROCK inhibitors has so far been limited by systemic or local ROCK-associated side effects. A soft drug approach, which involves predictable metabolic inactivation of an active compound to a nontoxic metabolite, could represent an attractive way to obtain ROCK inhibitors with improved tolerability. We herein report the design and synthesis of a new series of soft ROCK inhibitors structurally related to the ROCK inhibitor Y-27632. These inhibitors contain carboxylic ester moieties which allow inactivation by esterases. While the parent esters display strong activity in enzymatic (ROCK2) and cellular (MLC phosphorylation) assays, their corresponding carboxylic acid metabolites have negligible functional activity. Compound 32 combined strong efficacy (ROCK2 IC50 = 2.5 nM) with rapid inactivation in plasma (t1/2 <5′). Compound 32 also demonstrated in vivo efficacy when evaluated as an IOP-lowering agent in ocular normotensive New-Zealand White rabbits, without ocular side effects. 相似文献
19.
Ishichi Y Sasaki M Setoh M Tsukamoto T Miwatashi S Nagabukuro H Okanishi S Imai S Saikawa R Doi T Ishihara Y 《Bioorganic & medicinal chemistry》2005,13(6):1901-1911
As part of an on-going investigation to develop an increasing agent on rhythmic bladder contractions, 1-aryl-3-(1-benzylpiperidin-4-yl)propanones were synthesized and examined as noncarbamate acetylcholinesterase (AChE) inhibitors. Among compounds with various aryl groups, 1,2,5,6-tetrahydro-4H-pyrrolo[3,2,1-ij]quinolin-4-one derivative 9c was found to possess a potent AChE inhibition activity with an IC(50) value of 1.3nM. The compound 9c increased rhythmic bladder contractions in Guinea pigs and rats without affecting the basal intravesical pressure, which suggests that 9c may be useful for the treatment of voiding dysfunction caused by detrusor underactivity. 相似文献