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981.
The crystal structure of EcoRV endonuclease bound to non-cognate DNA at 2.0 angstroms resolution shows that very small structural adaptations are sufficient to ensure the extreme sequence specificity characteristic of restriction enzymes. EcoRV bends its specific GATATC site sharply by 50 degrees into the major groove at the center TA step, generating unusual base-base interactions along each individual DNA strand. In the symmetric non-cognate complex bound to GAATTC, the center step bend is relaxed to avoid steric hindrance caused by the different placement of the exocyclic thymine methyl groups. The decreased base-pair unstacking in turn leads to small conformational rearrangements in the sugar-phosphate backbone, sufficient to destabilize binding of crucial divalent metal ions in the active site. A second crystal structure of EcoRV bound to the base-analog GAAUTC site shows that the 50 degrees center-step bend of the DNA is restored. However, while divalent metals bind at high occupancy in this structure, one metal ion shifts away from binding at the scissile DNA phosphate to a position near the 3'-adjacent phosphate group. This may explain why the 10(4)-fold attenuated cleavage efficiency toward GAATTC is reconstituted by less than tenfold toward GAAUTC. Examination of DNA binding and bending by equilibrium and stopped-flow florescence quenching and fluorescence resonance energy transfer (FRET) methods demonstrates that the capacity of EcoRV to bend the GAATTC non-cognate site is severely limited, but that full bending of GAAUTC is achieved at only a threefold reduced rate compared with the cognate complex. Together, the structural and biochemical data demonstrate the existence of distinct mechanisms for ensuring specificity at the bending and catalytic steps, respectively. The limited conformational rearrangements observed in the EcoRV non-cognate complex provide a sharp contrast to the extensive structural changes found in a non-cognate BamHI-DNA crystal structure, thus demonstrating a diversity of mechanisms by which restriction enzymes are able to achieve specificity. 相似文献
982.
Relation of enzyme activity to local/global stability of murine adenosine deaminase: 19F NMR studies
Adenosine deaminase (ADA, EC 3.5.4.4) is a ubiquitous (beta/alpha)8-barrel enzyme crucial for purine metabolism and normal immune competence. In this study, it was observed that loss of enzyme activity of murine ADA (mADA) precedes the global secondary and tertiary structure transition when the protein is exposed to denaturant. The structural mechanism for this phenomenon was probed using site-specific 19F NMR spectroscopy in combination with [6-19F]tryptophan labeling and inhibitor binding. There are four tryptophan residues in mADA and all are located more than 12 A from the catalytic site. The 19F NMR spectra of [6-19F]Trp-labelled mADA show that the urea-induced chemical shift change of 19F resonance of W161, one of the four tryptophan 19F nuclei, correlates with the loss of enzyme activity. The urea-induced chemical shift change of another 19F resonance of W117 correlates with the change of the apparent rate constant for the binding of transition-state analogue inhibitor deoxycoformycin to the enzyme. On the other hand, the chemical environment of the local region around W264 does not change significantly, as a consequence of perturbation by low concentrations of urea or substrate analog. The results indicate that different regions of mADA have different local stability, which controls the activity and stability of the enzyme. The results provide new insights into the relationship between the function of a protein and its conformational flexibility as well as its global stability. This study illustrates the advantage of 19F NMR spectroscopy in probing site-related conformational change information in ligand binding, enzymatic activity and protein folding. 相似文献
983.
Gustafson CL Stauffacher CV Hallenga K Van Etten RL 《Protein science : a publication of the Protein Society》2005,14(10):2515-2525
Eukaryotic low-molecular-weight protein tyrosine phosphatases (LMW PTPs) contain a conserved serine, a histidine with an elevated pKa, and an active site asparagine that together form a highly conserved hydrogen bonding network. This network stabilizes the active site phosphate binding loop for optimal substrate binding and catalysis. In the phosphatase from the bovine parasite Tritrichomonas foetus (TPTP), both the conserved serine (S37) and asparagine (N14) are present, but the conserved histidine has been replaced by a glutamine residue (Q67). Site-directed mutagenesis, kinetic, and spectroscopic experiments suggest that Q67 is located near the active site and is important for optimal catalytic activity. Kinetic experiments also suggest that S37 participates in the active site/hydrogen bonding network. Nuclear magnetic resonance spectroscopy was used to determine the three-dimensional structure of the TPTP enzyme and to further examine the roles of S37 and Q67. The backbone conformation of the TPTP phosphate binding loop is nearly superimposable with that of other tyrosine phosphatases, with N14 existing in a strained, left-handed conformation that is a hallmark of the active site hydrogen bonding network in the LMW PTPs. As expected, both S37 and Q67 are located at the active site, but in the consensus structure they are not within hydrogen bonding distance of N14. The hydrogen bond interactions that are observed in X-ray structures of LMW PTPs may in fact be transient in solution. Protein dynamics within the active site hydrogen bonding network appear to be affected by the presence of substrate or bound inhibitors such as inorganic phosphate. 相似文献
984.
The cathepsin B-like proteinase from Helicoverpa armigera (HCB) is involved in the degradation of yolk proteins during embryonic development. In order to gain insight into the substrate
specificity of this proteinase, various proteins from animals and plants were tested as substrates. The specific cleavage
sites of this enzyme on endopeptide bonds were assayed using bovine serum albumin (BSA) as a substrate. Results showed that
BSA was degraded into several fragments, which suggests that HCB cleaves BSA at specific endopeptidyl sites. The amino acid
sequences of the BSA derived peptides were determined, revealing cleavage of the bonds between residues Arg81–Glu82, Val423–Glu424 and Gly430–Lys431. This suggests that the minimum requirement for a scissile bond to be recognized by HCB is the presence of an ionic amino
acid at the P1′ position and the P1 position can vary. These observations suggest that HCB cleaves bonds at the N-terminal side of ionic amino acid residues
giving HCB a wide range of substrates, though other factors dictating the substrate specificity of this enzyme remains to
be clarified. Our results provide new evidence that HCB functions as an endopeptidase on some proteins. 相似文献
985.
Rui Cai Yunmei Sun Naren Qimuge Guoqiang Wang Yingqian Wang Guiyan Chu Taiyong Yu Gongshe Yang Weijun Pang 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2018,1863(4):420-432
Adiponectin (AdipoQ) is an adipocyte-derived hormone with positive function on systemic glucose and lipid metabolism. Long noncoding RNA (lncRNA) is emerging as a vital regulator of adipogenesis. However, AdipoQ-related lncRNAs in lipid metabolism have not been explored. Here, AdipoQ antisense (AS) lncRNA was first identified, and we further found that it inhibited adipogenesis. The half-life of AdipoQ AS lncRNA was 10?h, whereas that of AdipoQ mRNA was 4?h. During adipogenic differentiation, AdipoQ AS lncRNA translocated from nucleus to cytoplasm. AdipoQ AS lncRNA and AdipoQ mRNA formed an RNA duplex. Moreover, AdipoQ AS lncRNA delivered via injection of adenovirus expressing AdipoQ AS lncRNA decreases white adipose tissue (WAT), brown adipose tissue (BAT) and liver triglycerides (TG) in mice consuming a high fat diet (HFD). Interestingly, the non-overlapping region of AdipoQ AS lncRNA improved serum glucose tolerance and insulin sensitivity in HFD mice, but not AdipoQ AS lncRNA. In conclusion, AdipoQ AS lncRNA transfer from nucleus to cytoplasm inhibits adipogenesis through formation of an AdipoQ AS lncRNA/AdipoQ mRNA duplex to suppress the translation of AdipoQ mRNA. Taken together, we suggest that AdipoQ AS lncRNA is a novel therapeutic target for obesity-related metabolic diseases. 相似文献
986.
Guoyuan Qi Rui Guo Haoyu Tian Lixia Li Hua Liu Yashi Mi Xuebo Liu 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2018,1863(6):549-562
Scope
Circadian clock plays a principal role in orchestrating our daily physiology and metabolism, and their perturbation can evoke metabolic diseases such as fatty liver and insulin resistance. Nobiletin (NOB) has been demonstrated to possess antitumor and neuroprotective activities. The objective of the current study is to determine potential effects of NOB on modulating the core clock gene Bmal1 regarding ameliorating glucolipid metabolic disorders.Results
Our results revealed that NOB partially reverse the relatively shallow daily oscillations of circadian clock genes and reset phase-shifting circadian rhythms in primary hepatocytes under metabolic disorders conditions. Importantly, NOB was found to be effective at amplifying glucose uptake via stimulating IRS-1/AKT signaling pathway, as well as blunting palmitate-induced lipogenesis in HepG2 cells via modulating AMPK-Sirt1 signaling pathway and key enzymes of de novo lipogenesis in a Bmal1-dependent manner. NOB attenuated palmitate-stimulated excessive secretions of ROS, restored the depletions of mitochondrial membrane potential, which is similar to the recovery in expressions of mitochondrial respiration complex I-IV.Conclusion
This study is the first to provide compelling evidences that NOB prevent cellular glucolipid metabolic imbalance and mitochondrial function in a Bmal1-dependent manner. Overall, NOB may serve as a nutritional preventive strategy in recovering metabolic disorders relevant to circadian clock. 相似文献987.
Distance,environmental and substrate factors impacting recovery of bryophyte communities after harvesting
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Thomas P. Baker Gregory J. Jordan Nicholas M. Fountain‐Jones Jayne Balmer Patrick J. Dalton Susan C. Baker 《应用植被学》2018,21(1):64-75
Aims
Bryophyte re‐colonization after disturbance is largely governed by environmental conditions within disturbed forests. In particular, distance to a forest edge is an important predictor of bryophyte community re‐colonization, through either direct constraints, such as dispersal limitation, or indirectly by altering environmental conditions. This study examines a range of factors – environmental, distance to an edge, substrate specific environment or local‐level environment – to determine which are important in the re‐colonization of bryophyte communities after forest harvesting. As bryophyte communities vary with the particular substrate inhabited, responses were examined across four substrates (rock, exposed roots, ground and CWD).Location
Tasmanian southern forests, Australia.Methods
Bryophyte composition was examined on four substrates (ground, coarse wood debris, exposed roots, rocks) within three ages (~7, ~27 and ~45 years post‐disturbance) of harvested wet eucalypt forest. Re‐colonization success of bryophyte communities was determined by comparing communities in regeneration forest to mature forest communities using axis scores from one‐dimensional constrained ordination. The importance of various environmental conditions for re‐colonization success was then modelled. Finally, path analysis was used to determine whether the impact of distance to a forest edge was meditated through its effects on key environmental variables.Results
Multiple environmental factors impacted re‐colonization of mature bryophyte communities. Local‐level conditions such as microclimate (temperature, humidity and VPD) and LAI were the most important in determining re‐colonization across substrates. Path analysis showed that distance to a forest edge had a significant impact on re‐colonization success, but only a relatively small part of this was mediated through its impact on environmental factors.Conclusions
Bryophyte re‐colonization is driven by a combination of microclimate conditions and factors related to distance from a forest edge (most likely dispersal distance). While some substrate‐specific factors impact bryophyte re‐colonization success, the consistent impact of local environmental factors across substrates suggests that harvesting management strategies that develop more ‘mature’ microclimate conditions and increase proximity to nearby mature forest patches will be beneficial for all bryophytes communities. As bryophyte re‐colonization was correlated with temporally dynamic environmental conditions, we suggest that forest age needs to be considered in future work. 相似文献988.
989.
Bioturbation and directionality in Earth's carbon isotope record across the Neoproterozoic–Cambrian transition
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Mixing of sediments by moving animals becomes apparent in the trace fossil record from about 550 million years ago (Ma), loosely overlapping with the tail end of the extreme carbonate carbon isotope δ13Ccarbonate fluctuations that qualitatively distinguish the Proterozoic geochemical record from that of the Phanerozoic. These Precambrian‐scale fluctuations in δ13Ccarbonate (PSF‐δ13Ccarbonate) remain enigmatic, due to their high amplitude and inclusion of global‐scale negative δ13Ccarbonate values, below anything attributable to mantle input. Here, we note that different biogeochemical‐model scenarios plausibly explaining globally synchronous PSF‐δ13Ccarbonate converge: via mechanistic requirements for extensive anoxia in marine sediments to support sedimentary build‐up of 13C‐depleted carbon. We hypothesize that bioturbation qualitatively reduced marine sediment anoxia by exposing sediments to oxygenated overlying waters, which ultimately contributed to decreasing the carbon cycle's subsequent susceptibility to PSF‐ δ13Ccarbonate. Bioturbation may also have reduced the quantity of (isotopically light) organic‐derived carbon available to contribute to PSF‐ δ13Ccarbonate via ocean crust carbonatization at depth. We conduct a comparative modelling exercise in which we introduce bioturbation to existing model scenarios for PSF‐ δ13Ccarbonate: expressing both the anoxic proportion of marine sediments, and the global organic carbon burial efficiency, as a decreasing function of bioturbation. We find that bioturbation's oxygenating impact on sediments has the capacity to prevent PSF‐ δ13Ccarbonate caused by authigenic carbonate precipitation or methanogenesis. Bioturbation's impact on the f‐ratio via remineralization is partially offset by liberation of organic phosphate, some of which feeds back into new production. We emphasize that this study is semiquantitative, exploratory and intended merely to provide a qualitative theoretical framework within which bioturbation's impact on long‐term, first‐order δ13Ccarbonate can be assessed (and it is hoped quantified in more detail by future work). With this proviso, we conclude that it is entirely plausible that bioturbation made a decisive contribution to the enigmatic directionality in the δ13Ccarbonate record, from the Neoproterozoic–Cambrian boundary onwards. 相似文献
990.
Ariane Wagner Francesca Di Bartolomeo Isabella Klein Claudia Hrastnik Kim Nguyen Doan Thomas Becker Günther Daum 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2018,1863(2):117-125
Phosphatidylserine decarboxylase 1 (Psd1p) catalyzes the formation of the majority of phosphatidylethanolamine (PE) in the yeast Saccharomyces cerevisiae. Psd1p is localized to mitochondria, anchored to the inner mitochondrial membrane (IMM) through membrane spanning domains and oriented towards the mitochondrial intermembrane space. We found that Psd1p harbors at least two inner membrane-associated domains, which we named IM1 and IM2. IM1 is important for proper orientation of Psd1p within the IMM (Horvath et al., J. Biol. Chem. 287 (2012) 36744–55), whereas it remained unclear whether IM2 is important for membrane-association of Psd1p. To discover the role of IM2 in Psd1p import, processing and assembly into the mitochondria, we constructed Psd1p variants with deletions in IM2. Removal of the complete IM2 led to an altered topology of the protein with the soluble domain exposed to the matrix and to decreased enzyme activity. Psd1p variants lacking portions of the N-terminal moiety of IM2 were inserted into IMM with an altered topology. Psd1p variants with deletions of C-terminal portions of IM2 accumulated at the outer mitochondrial membrane and lost their enzyme activity. In conclusion we showed that IM2 is essential for full enzymatic activity, maturation and correct integration of yeast Psd1p into the inner mitochondrial membrane. 相似文献