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991.
Investigation of Serine‐Proteinase‐Catalyzed Peptide Splicing in Analogues of Sunflower Trypsin Inhibitor 1 (SFTI‐1) 下载免费PDF全文
Natalia Karna Dr. Anna Łęgowska Dr. Stanisław Malicki Dr. Dawid Dębowski Przemysław Golik Agata Gitlin Dr. Przemysław Grudnik Dr. Benedykt Wladyka Dr. Krzysztof Brzozowski Dr. Grzegorz Dubin Prof. Krzysztof Rolka 《Chembiochem : a European journal of chemical biology》2015,16(14):2036-2045
Serine‐proteinase‐catalyzed peptide splicing was demonstrated in analogues of the trypsin inhibitor SFTI‐1: both single peptides and two‐peptide chains (C‐ and N‐terminal peptide chains linked by a disulfide bridge). In the second series, peptide splicing with catalytic amount of proteinase was observed only when formation of acyl–enzyme intermediate was preceded by hydrolysis of the substrate Lys–Ser peptide bond. Here we demonstrate that with an equimolar amount of the proteinase, splicing occurs in all the two‐peptide‐chain analogues. This conclusion was supported by high resolution crystal structures of selected analogues in complex with trypsin. We showed that the process followed a direct transpeptidation mechanism. Thus, the acyl–enzyme intermediate was formed and was immediately used for a new peptide bond formation; products associated with the hydrolysis of the acyl–enzyme were not observed. The peptide splicing was sequence‐ not structure‐specific. 相似文献
992.
Dr. Ryota Hidese Dr. Kenichi Ataka Dr. Eckhard Bill Dr. Seigo Shima 《Chembiochem : a European journal of chemical biology》2015,16(13):1861-1865
[Fe]‐Hydrogenase (Hmd) catalyzes reversible hydride transfer from H2. It harbors an iron‐guanylylpyridinol as a cofactor with an FeII that is ligated to one thiolate, two COs, one acyl‐C, one pyridinol‐N, and solvent. Here, we report that CuI and H2O2 inactivate Hmd (half‐maximal rates at 1 μM CuI and 20 μM H2O2) and that FeII inhibits the enzyme with very high affinity (Ki=40 nM ). Infrared and EPR studies together with competitive inhibition studies with isocyanide indicated that CuI exerts its inhibitory effect most probably by binding to the active site iron‐thiolate ligand. Using the same methods, it was found that H2O2 binds to the active‐site iron at the solvent‐binding site and oxidizes FeII to FeIII. Also it was shown that FeII reversibly binds away from the active site iron, with binding being competitive to the organic hydride acceptor; this inhibition is specific for FeII and is reminiscent of that for the [FeFe]‐hydrogenase second iron, which specifically interacts with H2. 相似文献
993.
Decoding the Chemical Language of Motile Bacteria by Using High‐Throughput Microfluidic Assays 下载免费PDF全文
John A. Crooks Matthew D. Stilwell Dr. Piercen M. Oliver Zhou Zhong Prof. Dr. Douglas B. Weibel 《Chembiochem : a European journal of chemical biology》2015,16(15):2151-2155
Motile bacteria navigate chemical environments by using chemoreceptors. The output of these protein sensors is linked to motility machinery and enables bacteria to follow chemical gradients. Understanding the chemical specificity of different families of chemoreceptors is essential for predicting and controlling bacterial behavior in ecological niches, including symbiotic and pathogenic interactions with plants and mammals. The identification of chemical(s) recognized by specific families of receptors is limited by the low throughput and complexity of chemotaxis assays. To address this challenge, we developed a microfluidic‐based chemotaxis assay that is quantitative, simple, and enables high‐throughput measurements of bacterial response to different chemicals. Using the model bacterium Escherichia coli, we demonstrated a strategy for identifying molecules that activate chemoreceptors from a diverse compound library and for determining how global behavioral strategies are tuned to chemical environments. 相似文献
994.
Investigating the Role of Conformational Effects on Laccase Stability and Hyperactivation under Stress Conditions 下载免费PDF全文
Dr. Valerio Ferrario Dr. Alexey Chernykh Federica Fiorindo Dr. Marina Kolomytseva Dr. Loris Sinigoi Dr. Nina Myasoedova Dr. Diana Fattor Prof. Dr. Cynthia Ebert Prof. Dr. Ludmila Golovleva Prof. Dr. Lucia Gardossi 《Chembiochem : a European journal of chemical biology》2015,16(16):2365-2372
Fungal laccase from Steccherinum ochraceum 1833 displays remarkable stability under different harsh conditions: organic/buffer mixtures, thermal treatment, and microwave radiation. The behavior is particularly significant in the light of the sharp inactivation observed for two different fungal laccases. Laccase from S. ochraceum 1833 also displays hyperactivation under mild thermal treatment (60 °C). Molecular dynamics simulations at 80 °C explained how this laccase retains the geometry of the electron transfer pathway, thereby assuring electron transfer through the copper ions and thus maintaining its catalytic activity at high temperature. Spectroscopic studies revealed that the thermal activation corresponds to specific conformational changes in the protein. The results indicate that this laccase is potentially applicable under denaturing conditions that might be beneficial for the biotransformation of recalcitrant substrates. 相似文献
995.
An Amphiphilic BODIPY‐Porphyrin Conjugate: Intense Two‐Photon Absorption and Rapid Cellular Uptake for Two‐Photon‐Induced Imaging and Photodynamic Therapy 下载免费PDF全文
Dr. Tao Zhang Dr. Rongfeng Lan Longlong Gong Dr. Baoyan Wu Yuzhi Wang Dr. Daniel W. J. Kwong Prof. Wai‐Kwok Wong Dr. Ka‐Leung Wong Prof. Da Xing 《Chembiochem : a European journal of chemical biology》2015,16(16):2357-2364
The new amphiphilic BODPY‐porphyrin conjugate BZnPP and its precursor BZnPH were synthesised, and their linear and two‐photon photophysical properties, together with their cellular uptake and photo‐cytotoxicity, were studied. This amphiphilic conjugate consists of a hydrophobic BODIPY moiety and a hydrophilic tetra(ethylene glycol) chain bridging a cationic triphenylphosphonium group to an amphiphilic porphyrin ZnP through acetylide linkers at its meso positions. A large two‐photon absorption cross‐section (σ=1725 GM) and a high singlet oxygen quantum yield (0.52) were recorded. Intense linear‐ and two‐photon‐induced red emissions were also observed for both BZnPP and BZnPH. Further in vitro studies showed that BZnPP exhibited very efficient cellular uptake and strong photocytotoxic but weak dark cytotoxic properties towards human breast carcinoma MCF‐7 cells. In summary, the two‐photon‐induced emission and the potent photo‐cytotoxicity of BZnPP make it an efficacious dual‐purpose tumour‐imaging and photodynamic therapeutic agent in the tissue‐transparent spectral windows. 相似文献
996.
Kuwanon‐L as a New Allosteric HIV‐1 Integrase Inhibitor: Molecular Modeling and Biological Evaluation 下载免费PDF全文
Dr. Francesca Esposito Dr. Cristina Tintori Dr. Riccardo Martini Dr. Frauke Christ Prof. Zeger Debyser Roberto Ferrarese Dr. Gianluigi Cabiddu Dr. Angela Corona Dr. Elisa Rita Ceresola Dr. Andrea Calcaterra Dr. Valentina Iovine Prof. Bruno Botta Dr. Massimo Clementi Dr. Filippo Canducci Prof. Maurizio Botta Prof. Enzo Tramontano 《Chembiochem : a European journal of chemical biology》2015,16(17):2507-2512
HIV‐1 integrase (IN) active site inhibitors are the latest class of drugs approved for HIV treatment. The selection of IN strand‐transfer drug‐resistant HIV strains in patients supports the development of new agents that are active as allosteric IN inhibitors. Here, a docking‐based virtual screening has been applied to a small library of natural ligands to identify new allosteric IN inhibitors that target the sucrose binding pocket. From theoretical studies, kuwanon‐L emerged as the most promising binder and was thus selected for biological studies. Biochemical studies showed that kuwanon‐L is able to inhibit the HIV‐1 IN catalytic activity in the absence and in the presence of LEDGF/p75 protein, the IN dimerization, and the IN/LEDGF binding. Kuwanon‐L also inhibited HIV‐1 replication in cell cultures. Overall, docking and biochemical results suggest that kuwanon‐L binds to an allosteric binding pocket and can be considered an attractive lead for the development of new allosteric IN antiviral agents. 相似文献
997.
Dr. Riccardo De Ricco Dr. Daniela Valensin Dr. Simone Dell'Acqua Prof. Dr. Luigi Casella Dr. Christelle Hureau Prof. Dr. Peter Faller 《Chembiochem : a European journal of chemical biology》2015,16(16):2319-2328
Copper binding to α‐synuclein (aS) and to amyloid‐β (Ab) has been connected to Parkinson's and Alzheimer's disease (AD), respectively, because Cu ions can modulate the peptide aggregation, and these Cu ? peptide complexes can catalyse the production of reactive oxygen species (ROS). In a significant proportion of AD brains, aggregation of aS and Ab has been detected, and it was proposed that Ab and aS interact with each other. Thus, we investigated the potential interactions of Ab and aS through their binding of copper(I) and copper(II). Additionally, β‐synuclein (bS) was investigated, due to its additional methionine residue, a potential CuI ligand. We found that: 1) the peptides containing the Cu‐binding domains Ab1–16, aS1–15 and bS1–15 have similar affinities towards CuII and towards CuI, with Ab1–16 being slightly stronger, 2) in the case of CuI, the additional Met residue in bS1–15 increased the affinity slightly, 3) the exchange of CuI/II between the two peptides is rapid (≤ms), 4) a/bS1–15 and Ab1–16 form a heterodimeric complex with CuII, 5) CuI probably promotes a transient ternary complex, 6) the different CuI/II coordination of Ab1–16, aS1–15 and bS1–15 impacts the capacity to produce ROS and to oxidise catechol, and 7) when Ab1–16, aS1–15 and Cu are present, the ROS production more closely resembles that by Ab1–16. The work gives insights into the coordination chemistry of these related peptides, and the relevance of coordination differences, the ternary complex and ROS production are discussed. 相似文献
998.
Comparison of 10,11‐Dehydrocurvularin Polyketide Synthases from Alternaria cinerariae and Aspergillus terreus Highlights Key Structural Motifs 下载免费PDF全文
Rachel V. K. Cochrane Dr. Zhizeng Gao Gareth R. Lambkin Dr. Wei Xu Prof. Jaclyn M. Winter Sandra L. Marcus Prof. Yi Tang Prof. John C. Vederas 《Chembiochem : a European journal of chemical biology》2015,16(17):2479-2483
Iterative type I polyketide synthases (PKSs) from fungi are multifunctional enzymes that use their active sites repeatedly in a highly ordered sequence to assemble complex natural products. A phytotoxic macrolide with anticancer properties, 10,11‐dehydrocurvularin (DHC), is produced by cooperation of a highly reducing (HR) iterative PKS and a non‐reducing (NR) iterative PKS. We have identified the DHC gene cluster in Alternaria cinerariae, heterologously expressed the active HR PKS (Dhc3) and NR PKS (Dhc5) in yeast, and compared them to corresponding proteins that make DHC in Aspergillus terreus. Phylogenetic analysis and homology modeling of these enzymes identified variable surfaces and conserved motifs that are implicated in product formation. 相似文献
999.
Phenylalanine Ammonia‐Lyase‐Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles 下载免费PDF全文
Diána Weiser Dr. László Csaba Bencze Gergely Bánóczi Ferenc Ender Dr. Róbert Kiss Eszter Kókai Dr. András Szilágyi Prof. Dr. Beáta G. Vértessy Dr. Ödön Farkas Dr. Csaba Paizs Prof. Dr. László Poppe 《Chembiochem : a European journal of chemical biology》2015,16(16):2283-2288
Phenylalanine ammonia‐lyase (PAL), found in many organisms, catalyzes the deamination of l ‐phenylalanine (Phe) to (E)‐cinnamate by the aid of its MIO prosthetic group. By using PAL immobilized on magnetic nanoparticles and fixed in a microfluidic reactor with an in‐line UV detector, we demonstrated that PAL can catalyze ammonia elimination from the acyclic propargylglycine (PG) to yield (E)‐pent‐2‐ene‐4‐ynoate. This highlights new opportunities to extend MIO enzymes towards acyclic substrates. As PG is acyclic, its deamination cannot involve a Friedel–Crafts‐type attack at an aromatic ring. The reversibility of the PAL reaction, demonstrated by the ammonia addition to (E)‐pent‐2‐ene‐4‐ynoate yielding enantiopure l ‐PG, contradicts the proposed highly exothermic single‐step mechanism. Computations with the QM/MM models of the N‐MIO intermediates from l ‐PG and l ‐Phe in PAL show similar arrangements within the active site, thus supporting a mechanism via the N‐MIO intermediate. 相似文献
1000.
A Multiple‐Labeling Strategy for Nonribosomal Peptide Synthetases Using Active‐Site‐Directed Proteomic Probes for Adenylation Domains 下载免费PDF全文
Dr. Fumihiro Ishikawa Dr. Takehiro Suzuki Dr. Naoshi Dohmae Prof. Hideaki Kakeya 《Chembiochem : a European journal of chemical biology》2015,16(18):2590-2594
Genetic approaches have greatly contributed to our understanding of nonribosomal peptide biosynthetic machinery; however, proteomic investigations are limited. Here, we developed a highly sensitive detection strategy for multidomain nonribosomal peptide synthetases (NRPSs) by using a multiple‐labeling technique with active‐site‐directed probes for adenylation domains. When applied to gramicidin S‐producing and ‐nonproducing strains of Aneurinibacillus migulanus (DSM 5759 and DSM 2895, respectively), the multiple technique sensitively detected an active multidomain NRPS (GrsB) in lysates obtained from the organisms. This functional proteomics method revealed an unknown inactive precursor (or other inactive form) of GrsB in the nonproducing strain. This method provides a new option for the direct detection, functional analysis, and high‐resolution identification of low‐abundance active NRPS enzymes in native proteomic environments. 相似文献