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Arredondo S Segatori L Gilbert HF Georgiou G 《The Journal of biological chemistry》2008,283(46):31469-31476
The Escherichia coli disulfide isomerase, DsbC is a V-shaped homodimer with each monomer comprising a dimerization region that forms part of a putative peptide-binding pocket and a thioredoxin catalytic domain. Disulfide isomerases from prokaryotes and eukaryotes exhibit little sequence homology but display very similar structural organization with two thioredoxin domains facing each other on top of the dimerization/peptide-binding region. To aid the understanding of the mechanistic significance of thioredoxin domain dimerization and of the peptide-binding cleft of DsbC, we constructed a series of protein chimeras comprising unrelated protein dimerization domains fused to thioredoxin superfamily enzymes. Chimeras consisting of the dimerization domain and the alpha-helical linker of the bacterial proline cis/trans isomerase FkpA and the periplasmic oxidase DsbA gave rise to enzymes that catalyzed the folding of multidisulfide substrate proteins in vivo with comparable efficiency to E. coli DsbC. In addition, expression of FkpA-DsbAs conferred modest resistance to CuCl2, a phenotype that depends on disulfide bond isomerization. Selection for resistance to elevated CuCl2 concentrations led to the isolation of FkpA-DsbA mutants containing a single amino acid substitution that changed the active site of the DsbA domain from CPHC into CPYC, increasing the similarity to the DsbC active site (CGYC). Unlike DsbC, which is resistant to oxidation by DsbB-DsbA and does not normally catalyze disulfide bond formation under physiological conditions, the FkpA-DsbA chimeras functioned both as oxidases and isomerases. The engineering of these efficient artificial isomerases delineates the key features of catalysis of disulfide bond isomerization and enhances our understanding of its evolution. 相似文献
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Reduction-reoxidation cycles contribute to catalysis of disulfide isomerization by protein-disulfide isomerase 总被引:1,自引:0,他引:1
Protein-disulfide isomerase (PDI) catalyzes the formation and isomerization of disulfides during oxidative protein folding. This process can be error-prone in its early stages, and any incorrect disulfides that form must be rearranged to their native configuration. When the second cysteine (CGHC) in the PDI active site is mutated to Ser, the isomerase activity drops by 7-8-fold, and a covalent intermediate with the substrate accumulates. This led to the proposal that the second active site cysteine provides an escape mechanism, preventing PDI from becoming trapped with substrates that isomerize slowly (Walker, K. W., and Gilbert, H. F. (1997) J. Biol. Chem. 272, 8845-8848). Escape also reduces the substrate, and if it is invoked frequently, disulfide isomerization will involve cycles of reduction and reoxidation in preference to intramolecular isomerization of the PDI-bound substrate. Using a gel-shift assay that adds a polyethylene glycol-conjugated maleimide of 5 kDa for each sulfhydryl group, we find that PDI reduction and oxidation are kinetically competent and essential for isomerization. Oxidants inhibit isomerization and oxidize PDI when a redox buffer is not present to maintain the PDI redox state. Reductants also inhibit isomerization as they deplete oxidized PDI. These rapid cycles of PDI oxidation and reduction suggest that PDI catalyzes isomerization by trial and error, reducing disulfides and oxidizing them in a different configuration. Disulfide reduction-reoxidation may set up critical folding intermediates for intramolecular isomerization, or it may serve as the only isomerization mechanism. In the absence of a redox buffer, these steady-state reduction-oxidation cycles can balance the redox state of PDI and support effective catalysis of disulfide isomerization. 相似文献
45.
Beltrán HI Damian-Zea C Hernández-Ortega S Nieto-Camacho A Ramírez-Apan MT 《Journal of inorganic biochemistry》2007,101(7):1070-1085
The one pot reactions carried among salicylaldehyde 1, ortho-aminophenols 2a-2g, and di-phenyl-tin(IV) oxide 3 led to seven di-phenyl-tin(IV) compounds 4a-4g in good yields (97-83%). All compounds were analyzed by IR, 1H, 13C, 119Sn NMR spectroscopy, mass spectrometry and elemental analyses; furthermore, in the case of compounds 4b, 4c, 4e and 4g by X-ray diffraction. Compounds 4a-4g were tested in vitro against six human tumor cell lines U251, PC-3, K-562, HCT-15, MCF-7 and SKLU-1 to assess their in vitro antitumor activity. The results suggest biological specificity towards U251, MCF-7 and SKLU-1 cells at doses below 2.5 microM, which are lower than cis-platin IC50's in the three cell lines. Since the inhibitory concentration values for the series were alike to Ph(2)SnCl(2) is feasible that only the Ph(2)Sn moiety is responsible for those activities, further experiments are under research. Besides, 4a-4g were tested for their antioxidant efficiency in rat brain homogenate showing that 4g is more active (IC50=3.01 microM) than the flavone quercetin (natural antioxidant, IC50=4.11 microM) on inhibition of thiobarbituric acid reactive substances (TBARS). The TBARS activity (IC50) correlates with the ortho-aminophenol substitutions and a linear combination among sigma Hammett, one bond tin coupling constants and tin chemical shifts against the measured IC(50-TBARS) was found. This correlation gave basis that the implied molecular variables can become trackers for the calculation of TBARS inhibitory concentrations in similar systems. Moreover, there seemed to be an inverse structure-response behavior among activities, since the 4g derivative is the less active compound for cytotoxic assays meanwhile it is the best in antioxidant tests. 相似文献
46.
Hiram E. Miller 《The Western journal of medicine》1930,33(3):662-663
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Rábago-Quiroz CH López-Martínez J Valdez-Holguín JE Nevárez Martínez MO 《Revista de biología tropical》2011,59(1):255-267
The Gulf of California is one of the most mega-diverse regions in the world, for which few fishery information is available. We present here latitudinal and bathymetric distribution of the most abundant and frequent bycatch species from the Gulf of California. The samples were obtained from a total of 111 hauls taken during seven research cruises of the closed shrimp season (2002-2005-2007), and also, from research cruises made at depths up to 90 m. Due to the high variety species in this experimental shrimp bycatch, only those with highest biological value index (BVI) were selected. A total of fifteen species had the highest BVI and represented about 60% of the total abundance. A total of 16 508 organisms were analyzed, representing 243 fish, crustacean, mollusk and echinoderm species. Fish were the most abundant, being the most frequent species: Urobatis halleri, Synodus scituliceps, Diplectrum pacificum, Haemulopsis nitidus and Eucinostomus argenteus. A wide latitudinal distribution of these species along the study area, as well as a bathymetric distribution from 9 to 67 m depth, was observed. Two of these species were found at 325 m depth. Due to the wide bathymetric distribution obtained, total abundances and sizes for each species by depth strata should be determined, and one can assume that deeper than 25 m, the capture of these species decreases, and these areas can be used as natural repopulation areas, for depths where they are mainly captured by the commercial shrimp fishery. 相似文献