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1.
Chemosphere ; 239: 124769, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31526997

RESUMO

In this study, Cerium chloride-induced conformational changes of Bovine Liver Catalase (BLC) has been investigated by molecular docking and further supported by various biophysical techniques. The temporal change of catalytic activity of BLC has also been studied in presence of Ce(III) with different buffer solution in vitro at 25 °C. The differential binding of Ce(III) to BLC observed by simulation study was well supported by the differential regulation of BLC activity in different buffers. After 1 h of incubation with CeCl3, the reduction in activity of BLC was maximum in MOPS, HEPES and Tris buffer, whereas no change in activity was noticed in phosphate buffer. Isothermal Titration Calorimetric (ITC) study also supports the differential binding of Ce(III) to BLC in different buffers. Ce(III)-induced conformational transition in BLC was followed as a function of concentration. Nevertheless, with 24 h incubation of CeCl3 the activity of BLC was highest with higher molar concentration of CeCl3 suggesting the conformational stability of BLC in presence of Ce(III). The compromised activity of BLC in response to Ce(III) is due to the induced conformational change and the degree of change in secondary conformation of BLC was maximum in MOPS, HEPES and Tris and least in phosphate buffer. Therefore, the reduced activity of BLC is controlled by the direct interaction of Ce(III) in the active site of BLC in Tris buffer or indirect interaction of Ce(III) in the non-active site of BLC in MOPS and HEPES buffer.


Assuntos
Catalase/química , Catalase/metabolismo , Cério/química , Fígado/enzimologia , Animais , Tampões (Química) , Calorimetria , Domínio Catalítico , Bovinos , Cério/metabolismo , Cloretos/química , Simulação de Acoplamento Molecular , Conformação Proteica
2.
Phys Chem Chem Phys ; 21(42): 23501-23513, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31617551

RESUMO

YIV-906 (formally PHY906, KD018) is a four-herb formulation that is currently being developed to improve the therapeutic index and ameliorate the side effects of many chemotherapeutic drugs including sorafenib, irinotecan, and capecitabine. However, as a promising anti-cancer adjuvant, the molecular mechanism of action of YIV-906 remains unrevealed due to its multi-component and multi-target features. Since YIV-906 has been shown to induce apoptosis and autophagy in cancer cells through modulating the negative regulators of ERK1/2, namely DUSPs, it is of great interest to elucidate the key components that cause the therapeutic effect of YIV-906. In this work, we investigated the mechanism of YIV-906 inhibiting DUSPs, using a broad spectrum of molecular modelling techniques, including molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations. In total, MD simulations and binding free energy calculations were performed for 99 DUSP-ligand complexes. We found that some herbal components or their metabolites could inhibit DUSPs. Based on the docking scores and binding free energies, the sulfation and glucuronidation metabolites of the S ingredient in YIV-906 play a leading role in inhibiting DUSPs, although several original herbal chemicals with carboxyl groups from the P and Z ingredients also make contributions to this inhibitory effect. It is not a surprise that the electrostatic interaction plays the dominant role in the ligand binding process, given the fact that several charged residues reside in the binding pockets of DUSPs. Our MD simulation results demonstrate that the sulfate moieties and carboxyl moieties of the advantageous ligands from YIV-906 can occupy the enzymes' catalytic sites, mimicking the endogenous phosphate substrates of DUSPs. As such, the ligand binding can inhibit the association of DUSPs and ERK1/2, which in turn reduces the dephosphorylation of ERK1/2 and causes cell cycle arrest in the tumor. Our modelling study provides useful insights into the rational design of highly potent anti-cancer drugs targeting DUSPs. Finally, we have demonstrated that multi-scale molecular modelling techniques are able to elucidate molecular mechanisms involving complex molecular systems.


Assuntos
Antineoplásicos Fitogênicos/química , Medicamentos de Ervas Chinesas/química , Antineoplásicos Fitogênicos/metabolismo , Sítios de Ligação , Domínio Catalítico , Medicamentos de Ervas Chinesas/metabolismo , Fosfatases de Especificidade Dupla/antagonistas & inibidores , Fosfatases de Especificidade Dupla/metabolismo , Humanos , Ligantes , Proteína Quinase 3 Ativada por Mitógeno/química , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Termodinâmica
3.
Inorg Chem ; 58(20): 13933-13944, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31566371

RESUMO

Density functional vibrational frequency calculations have been performed on eight geometry optimized cytochrome c oxidase (CcO) dinuclear center (DNC) reaction cycle intermediates and on the oxymyoglobin (oxyMb) active site. The calculated Fe-O and O-O stretching modes and their frequency shifts along the reaction cycle have been compared with the available resonance Raman (rR) measurements. The calculations support the proposal that in state A[Fea33+-O2-•···CuB+] of CcO, O2 binds with Fea32+ in a similar bent end-on geometry to that in oxyMb. The calculations show that the observed 20 cm-1 shift of the Fea3-O stretching mode from the PR to F state is caused by the protonation of the OH- ligand on CuB2+ (PR[Fea34+═O2-···HO--CuB2+] → F[Fea34+═O2-···H2O-CuB2+]), and that the H2O ligand is still on the CuB2+ site in the rR identified F[Fea34+═O2-···H2O-CuB2+] state. Further, the observed rR band at 356 cm-1 between states PR and F is likely an O-Fea3-porphyrin bending mode. The observed 450 cm-1 low Fea3-O frequency mode for the OH active oxidized state has been reproduced by our calculations on a nearly symmetrically bridged Fea33+-OH-CuB2+ structure with a relatively long Fea3-O distance near 2 Å. Based on Badger's rule, the calculated Fea3-O distances correlate well with the calculated νFe-O-2/3 (νFe-O is the Fea3-O stretching frequency) with correlation coefficient R = 0.973.


Assuntos
Teoria da Densidade Funcional , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Ferro/química , Oxigênio/química , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Complexo IV da Cadeia de Transporte de Elétrons/química , Ferro/metabolismo , Ligantes , Modelos Moleculares , Oxigênio/metabolismo , Vibração
4.
Phys Chem Chem Phys ; 21(39): 22103-22112, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31570909

RESUMO

Type III phosphatidylinositol 4 kinases (PI4KIIIs) are essential enzymes that are related to the replication of multiple RNA viruses. Understanding the interaction mechanisms of molecular compounds with the alpha and beta isoforms of PI4KIII (PI4KIIIα and PI4KIIIß) is of significance in the development of inhibitors that can bind to these two enzymes selectively. In this work, molecular dynamics (MD) simulations and binding free energy calculations were combined to investigate the binding modes of seven selected compounds to PI4KIIIα and PI4KIIIß. Analyses based on MD trajectories provide detailed interaction mechanisms of these compounds with PI4KIIIα and PI4KIIIß at the atomic level, and indicate that the selectivity of these compounds is mainly due to the structural difference of the binding pockets. It is expected that the detailed binding information found in this study can provide useful help for the structure-based design of selective inhibitors toward PI4KIIIα and PI4KIIIß.


Assuntos
1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/química , Sequência de Aminoácidos , Domínio Catalítico , Concentração Inibidora 50 , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Transdução de Sinais , Relação Estrutura-Atividade , Termodinâmica
5.
Phys Chem Chem Phys ; 21(42): 23408-23417, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31625550

RESUMO

In the field of artificial metalloenzyme (ArM) catalysis, how to identify the critical factors affecting the catalytic activity and enantioselectivity remains a challenge. In this work, the mechanism of enantioselective reduction of imine catalyzed by using [Rh(Me4Cpbiot)Cl2]·S112H Sav (denoted as S112H) and [Rh(Me4Cpbiot)Cl2]·K121H Sav (denoted as K121H) was studied by using molecular dynamics (MD) simulations combined with density functional theory (DFT) calculations. Four binding modes of imine, two proton sources (hydronium ion and lysine) and eight proposed reaction pathways were systematically discussed. The results showed that due to the anchoring effect of the mutation site of ArMs, the rhodium complex which oscillated like a pendulum was bound to a specific conformation, which further determined the chirality of the reduced product. C-Hπ, cation-π and ππ weak interactions played an important role in imine binding, and the favorable binding mode of imine was catalyzed by S112H in landscape orientation and catalyzed by K121H in portrait orientation, respectively. LYS121 is the most possible proton source in the S112H catalytic process while the proton source in the K121H catalytic process is the hydronium ion of the active sites. Furthermore, based on the reaction mechanism, modification of Rh(Me4Cpbiot)Cl2 was carried out in S112H and K121H, and the results suggested that the reaction barrier could be effectively reduced by replacing the methyl groups on Cp* with an amino group. This work gives a fundamental understanding of the mechanism of ArMs toward the imine reduction reaction, in the hope of providing a strategy for reasonable designs of ArMs with high enantioselectivity.


Assuntos
Complexos de Coordenação/química , Iminas/química , Sítios de Ligação , Catálise , Domínio Catalítico , Complexos de Coordenação/metabolismo , Teoria da Densidade Funcional , Metaloproteínas/química , Metaloproteínas/metabolismo , Simulação de Dinâmica Molecular , Oxirredução , Ródio/química , Estereoisomerismo , Termodinâmica
6.
Nucleic Acids Res ; 47(18): 9761-9776, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31504772

RESUMO

Modification dependent restriction endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing domains into several groups. The TspA15I (VcaM4I, CmeDI), ScoA3IV (MsiJI, VcaCI) and YenY4I groups, all featuring a PUA superfamily domain, preferentially cleaved DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. ScoA3V, also featuring a PUA superfamily domain, but of a different clade, exhibited 6-methyladenine stimulated nicking activity. With few exceptions, ORFs for PUA-superfamily domain containing endonucleases were not close to DNA methyltransferase ORFs, strongly supporting modification dependent activity of the endonucleases. DUF3427 domain containing fusion proteins had very little or no endonuclease activity, despite the presence of a putative PD-(D/E)XK catalytic domain. However, their expression potently restricted phage T4gt in Escherichia coli cells. In contrast to the ORFs for PUA domain containing endonucleases, the ORFs for DUF3427 fusion proteins were frequently found in defense islands, often also featuring DNA methyltransferases.


Assuntos
Metilases de Modificação do DNA/genética , Enzimas de Restrição do DNA/genética , Escherichia coli/enzimologia , Regulação Enzimológica da Expressão Gênica/genética , Sequência de Aminoácidos , Domínio Catalítico/genética , Clivagem do DNA , Metilases de Modificação do DNA/química , Enzimas de Restrição do DNA/química , Enzimas de Restrição do DNA/classificação , Escherichia coli/genética , Modelos Moleculares , Estrutura Terciária de Proteína/genética , Alinhamento de Sequência
7.
Phys Chem Chem Phys ; 21(38): 21370-21380, 2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31531468

RESUMO

Density functional theory modelling has been used to design Mo and W-based catalysts MoIII(tBu)(CO) and WIII(tBu)(CO) for CO2 hydrogenation and HCOOH dehydrogenation, which are bio-mimics of the active site of formate dehydrogenase. Based on DFT calculations, the molybdenum and tungsten based complexes are good catalysts in the +3 oxidation state for CO2 hydrogenation with free energies of 24.03 and 21.31 kcal mol-1, respectively. Such a low barrier indicates that our newly designed Mo and W-based complexes are very efficient for CO2 hydrogenation or HCOOH dehydrogenation catalysis. Overall, our computational results provide in depth insights that can serve as a great tool for the design and development of new and efficient molybdenum and tungsten based catalysts for CO2 hydrogenation or HCOOH dehydrogenation.


Assuntos
Materiais Biomiméticos/química , Dióxido de Carbono/química , Complexos de Coordenação/química , Formiato Desidrogenases/química , Formiatos/química , Modelos Moleculares , Catálise , Domínio Catalítico , Hidrogenação , Ligantes , Molibdênio/química , Oxirredução , Termodinâmica , Tungstênio/química
8.
J Chem Theory Comput ; 15(11): 6074-6084, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31518121

RESUMO

Spin-dependent processes involving nonadiabatic transitions between electronic states with different spin multiplicities play important roles in the chemistry of complex systems. The rates of these processes can be predicted based on the molecular properties at the minimum energy crossing point (MECP) between electronic states. We present the development of the MECP search technique within the fragment molecular orbital (FMO) method applicable to large complex systems. The accuracy and scalability of the new method is demonstrated on several models of the metal-sulfur protein rubredoxin. The effect of the model size on the MECP geometry and relative energy is discussed. The fragment energy decomposition and spin density delocalization analyses reveal how different protein residues and solvent molecules contribute to stabilization of the spin states. The developed FMO-MECP method can help to clarify the role of nonadiabatic spin-dependent processes in complex systems and can be used for designing mutations aimed at controlling these processes in metalloproteins, including spin-dependent catalysis and electron transfer.


Assuntos
Modelos Moleculares , Teoria Quântica , Domínio Catalítico , Transporte de Elétrons , Rubredoxinas/química , Rubredoxinas/metabolismo , Termodinâmica
9.
J Agric Food Chem ; 67(40): 11158-11166, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31537069

RESUMO

There are ongoing interests in improving the galactooligosaccharide (GOS) synthesis efficiency of ß-galactosidase by protein engineering. In this study, an intelligent double-hydrophobic amino acid scanning strategy was proposed and employed to target nine residues forming the glycon-binding site (-1 subsite) of ß-galactosidase Bgal1-3. Two mutants C510V and H512I with significantly improved GOS synthesis efficiency were obtained. When 40% (w/v) lactose was used as a substrate, Bgal1-3 reached a maximum GOS yield of 45.3% at 16 h, while the mutants reached higher yields in a much shorter time (59.1% at 10 h for C510V, 51.5% at 2 h for H512I). When skim milk was treated with these enzymes, more GOS was produced (19.9 g/L for C510V, 12.7 g/L for H512I) than that for Bgal1-3 (10.3 g/L) at a lactose conversion of 90%. These results validated hydrophobicity scanning as an efficient method to engineer ß-galactosidases into promising catalysts for the preparation of GOS and GOS-enriched milk.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Galactose/química , Oligossacarídeos/química , beta-Galactosidase/química , Sequência de Aminoácidos , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Galactose/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lactose/química , Oligossacarídeos/metabolismo , Engenharia de Proteínas , Alinhamento de Sequência , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
10.
J Chem Theory Comput ; 15(11): 5894-5907, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31538779

RESUMO

A nonempirical quantum mechanical method for the efficient and accurate quantification and analysis of intermolecular interactions is presented and tested on existing benchmark sets. The leading idea here is to focus on the intermolecular part of the correlation energy that contains the all-important London dispersion (LD) interaction. To keep the cost of the method low, essentially at the level of a Hartree-Fock (HF) calculation, the intramolecular part of the correlation energy is neglected. We also neglect the nondispersive parts of the intermolecular correlation energy. This scheme that we denote as Hartree-Fock plus London dispersion (HFLD) can be readily realized on the basis of the recently reported multilevel implementation of the domain-based local pair natural orbital coupled-cluster (DLPNO-CC) theory in conjunction with the well-established local energy decomposition (LED) analysis. The accuracy and efficiency of the HFLD method are evaluated on rare gas dimers, on the S66 and L7 benchmark sets of noncovalent interactions, and on an additional set (LP14) consisting of bulky Lewis pairs held together by intermolecular interactions of various strengths, with interaction energies ranging from -8 to -107 kcal/mol. It is first shown that the LD energy calculated with this approach is essentially identical to that obtained from the full DLPNO-CCSD(T)/LED calculation, with a mean absolute error of 0.2 kcal/mol on the S66 benchmark set. Moreover, in terms of the overall interaction energies, the HFLD method shows an efficiency that is comparable to that of the HF method, while retaining an accuracy between that of the DLPNO-CCSD and DLPNO-CCSD(T) schemes. Since the underlying DLPNO-CCSD method is linear scaling with respect to the system size, the HFLD approach also does not lead to new bottlenecks for large systems. As an illustrative example of its efficiency, the HFLD scheme was applied to the interaction between the substrate and the residues in the active site of the cyclohexanone monooxygenase enzyme. The excellent cost/performance ratio indicates that the HFLD method opens new avenues for the accurate calculation and analysis of noncovalent interaction energies in large molecular systems.


Assuntos
Modelos Moleculares , Teoria Quântica , Domínio Catalítico , Dimerização , Gases/química , Oxigenases/química , Oxigenases/metabolismo , Especificidade por Substrato , Termodinâmica
11.
Inorg Chem ; 58(19): 12601-12608, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31539235

RESUMO

The radical SAM enzyme HydG generates CO- and CN--containing Fe complexes that are involved in the bioassembly of the [FeFe] hydrogenase active cofactor, the H-cluster. HydG contains a unique 5Fe-4S cluster in which the fifth "dangler" Fe and the coordinating cysteine molecule have both been shown to be essential for its function. Here, we demonstrate that this dangler Fe can be replaced with Ni2+ or Co2+ and that the cysteine can be replaced with selenocysteine. The resulting HydG variants were characterized by electron paramagnetic resonance and X-ray absorption spectroscopy, as well as subjected to a Tyr cleavage assay. Both Ni2+ and Co2+ are shown to be exchange-coupled to the 4Fe-4S cluster, and selenocysteine substitution does not alter the electronic structure significantly. XAS data provide details of the coordination environments near the Ni, Co, and Se atoms and support a close interaction of the dangler metal with the FeS cluster via an asymmetric SeCys bridge. Finally, while we were unable to observe the formation of novel organometallic species for the Ni2+ and Co2+ variants, the selenocysteine variant retains the activity of wild type HydG in forming [Fe(CO)x(CN)y] species. Our results provide more insights into the unique auxiliary cluster in HydG and expand the scope of artificially generated Fe-S clusters with heteroatoms.


Assuntos
Proteínas de Bactérias/química , Cobalto/química , Proteínas com Ferro-Enxofre/química , Níquel/química , Selenocisteína/química , Shewanella/química , Domínio Catalítico , Cisteína/química
12.
Chemistry ; 25(64): 14613-14624, 2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31498478

RESUMO

Histamine H4 receptor (H4 R) orthologues are G-protein-coupled receptors (GPCRs) that exhibit species-dependent basal activity. In contrast to the basally inactive mouse H4 R (mH4 R), human H4 R (hH4 R) shows a high degree of basal activity. We have performed long-timescale molecular dynamics simulations and rigidity analyses on wild-type hH4 R, the experimentally characterized hH4 R variants S179M, F169V, F169V+S179M, F168A, and on mH4 R to investigate the molecular nature of the differential basal activity. H4 R variant-dependent differences between essential motifs of GPCR activation and structural stabilities correlate with experimentally determined basal activities and provide a molecular explanation for the differences in basal activation. Strikingly, during the MD simulations, F16945.55 dips into the orthosteric binding pocket only in the case of hH4 R, thus adopting the role of an agonist and contributing to the stabilization of the active state. The results shed new light on the molecular mechanism of basal H4 R activation that are of importance for other GPCRs.


Assuntos
Fenilalanina/análogos & derivados , Receptores Histamínicos H4/agonistas , Animais , Sítios de Ligação , Domínio Catalítico , Humanos , Camundongos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Fenilalanina/química , Estabilidade Proteica , Receptores Histamínicos H4/genética , Receptores Histamínicos H4/metabolismo
13.
Eur J Med Chem ; 181: 111580, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31400708

RESUMO

A series of peptidomimetic compounds incorporating an electrophilic moiety was synthesized using the Ugi reaction. These compounds (termed the Ugi Michael acceptors or UMAs) were designed to target the selenocysteine catalytic residue of thioredoxin reductase 1 (TrxR1), a promising cancer target. The compounds were assessed for their potential to inhibit TrxR1 using human neuroblastoma (SH-SY5Y) cell lysate. Based on this initial screening, six compounds were selected for testing against recombinant rat TrxR1 and in the insulin assay to reveal low-micromolar to submicromolar potency of these inhibitors. The same frontrunner compounds were evaluated for their ability to exert antiproliferative activity and induce cell death and this activity was compared to the UMA effects on the levels of reactive oxygen and nitrogen species (RONS). Collectively, the UMA compounds class presented itself as a rich source of leads for TrxR1 inhibitor discovery for anticancer application. Compound 7 (DVD-445) was nominated a lead for further optimization.


Assuntos
Amidas/farmacologia , Antineoplásicos/farmacologia , Tiorredoxina Redutase 1/antagonistas & inibidores , Tiorredoxinas/metabolismo , Amidas/química , Antineoplásicos/química , Domínio Catalítico/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/metabolismo , Tiorredoxina Redutase 1/química , Tiorredoxina Redutase 1/metabolismo
14.
J Enzyme Inhib Med Chem ; 34(1): 1400-1413, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31401897

RESUMO

A large library of derivatives based on the scaffold of 2-(benzylsulfinyl)benzoic acid were synthesised and tested as atypical inhibitors against four different isoforms of human carbonic anhydrase (hCA I, II, IX and XII, EC 4.2.1.1). The exploration of the chemical space around the main functional groups led to the discovery of selective hCA IX inhibitors in the micromolar/nanomolar range, thus establishing robust structure-activity relationships within this versatile scaffold. HPLC separation of some selected chiral compounds and biological evaluation of the corresponding enantiomers was performed along with molecular modelling studies on the most active derivatives.


Assuntos
Ácido Benzoico/química , Inibidores da Anidrase Carbônica/química , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/efeitos dos fármacos , Desenho de Drogas , Isoenzimas/efeitos dos fármacos , Inibidores da Anidrase Carbônica/síntese química , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Humanos , Simulação de Acoplamento Molecular , Estereoisomerismo , Relação Estrutura-Atividade
15.
Nat Commun ; 10(1): 3435, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387991

RESUMO

Histones, the principal protein components of chromatin, contain long disordered sequences, which are extensively post-translationally modified. Although histone chaperones are known to control both the activity and specificity of histone-modifying enzymes, the mechanisms promoting modification of highly disordered substrates, such as lysine-acetylation within the N-terminal tail of histone H3, are not understood. Here, to understand how histone chaperones Asf1 and Vps75 together promote H3 K9-acetylation, we establish the solution structural model of the acetyltransferase Rtt109 in complex with Asf1 and Vps75 and the histone dimer H3:H4. We show that Vps75 promotes K9-acetylation by engaging the H3 N-terminal tail in fuzzy electrostatic interactions with its disordered C-terminal domain, thereby confining the H3 tail to a wide central cavity faced by the Rtt109 active site. These fuzzy interactions between disordered domains achieve localization of lysine residues in the H3 tail to the catalytic site with minimal loss of entropy, and may represent a common mechanism of enzymatic reactions involving highly disordered substrates.


Assuntos
Histona Acetiltransferases/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Acetilação , Domínio Catalítico , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Histona Acetiltransferases/isolamento & purificação , Chaperonas de Histonas/isolamento & purificação , Histonas/isolamento & purificação , Lisina/metabolismo , Chaperonas Moleculares/isolamento & purificação , Chaperonas Moleculares/metabolismo , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Proteínas de Xenopus/isolamento & purificação , Proteínas de Xenopus/metabolismo
16.
Chemistry ; 25(49): 11416-11421, 2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31407832

RESUMO

Trypanothione reductase (TR) plays a key role in the unique redox metabolism of trypanosomatids, the causative agents of human African trypanosomiasis (HAT), Chagas' disease, and leishmaniases. Introduction of a new, lean propargylic vector to a known class of TR inhibitors resulted in the strongest reported competitive inhibitor of Trypanosoma (T.) brucei TR, with an inhibition constant Ki of 73 nm, which is fully selective against human glutathione reductase (hGR). The best ligands exhibited in vitro IC50 values (half-maximal inhibitory concentration) against the HAT pathogen, T. brucei rhodesiense, in the mid-nanomolar range, reaching down to 50 nm. X-Ray co-crystal structures confirmed the binding mode of the ligands and revealed the presence of a HEPES buffer molecule in the large active site. Extension of the propargylic vector, guided by structure-based design, to replace the HEPES buffer molecule should give inhibitors with low nanomolar Ki and IC50 values for in vivo studies.


Assuntos
Desenho de Drogas , Inibidores Enzimáticos/química , NADH NADPH Oxirredutases/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Trypanosoma brucei brucei/enzimologia , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Concentração Inibidora 50 , Ligantes , Simulação de Dinâmica Molecular , NADH NADPH Oxirredutases/metabolismo , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade
17.
Artigo em Inglês | MEDLINE | ID: mdl-31454681

RESUMO

Clip domain serine proteases (CDSPs) participate in the extracellular signaling cascades of various biological processes such as innate immune responses in invertebrates. CDSP genes have been isolated from numerous invertebrates. Nevertheless, the enzymatic properties of mollusk CDSPs are poorly understood. In the present study, we demonstrated that the amino acid sequences of the trypsin-like serine protease purified from the digestive fluid of the sea hare, Aplysia kurodai resemble those of the unidentified CDSP-type protein (TPS3) of Aplysia californica predicted by genome analysis. The purified enzyme produced single 34 and 26.5 kDa bands on SDS-PAGE under non-reducing and reducing conditions, respectively. The 34-kDa band generated two amino-terminal sequences that were similar to the deduced sequences of the clip and catalytic domains of TPS3. The single amino-terminal sequence of the 26.5 kDa band showed a single sequence homologous to the catalytic domain. Thus, the purified enzyme consists of clip and catalytic domains bridged by disulfide linkage(s). The subsite specificity and inhibitor sensitivity of the purified enzyme were clearly distinct from those of horseshoe crab and silkworm CDSPs. A good substrate for the sea hare enzyme was pyroglutamyl-Arg-Thr-Lys-Arg-4-methyl-7-coumarylamide. The enzyme activity was strongly inhibited by aprotinin but not leupeptin. The physiological function of the enzyme in the digestive fluid remains to be determined.


Assuntos
Aplysia/enzimologia , Sistema Digestório/enzimologia , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Animais , Aplysia/genética , Domínio Catalítico , Eletroforese em Gel de Poliacrilamida , Serina Endopeptidases/genética , Especificidade por Substrato
18.
Chemistry ; 25(58): 13285-13289, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31441974

RESUMO

A calix[4]arene ligand, in which two of the phenol functions are replaced by pyrazole units has been employed to mimic the His2 -Tyr2 (His: histidine, Tyr: tyrosine) ligand sphere within the active site of the galactose oxidase (GO). The calixarene backbone forces the corresponding copper(II) complex into a see-saw-type structure, which is hitherto unprecedented in GO modelling chemistry. It undergoes a one-electron oxidation that is centered at the phenolate donor leading to a copper-coordinated phenoxyl radical like in the GO. Accordingly, the complex was tested as a functional model and indeed proved capable of oxidizing benzyl alcohol to the respective aldehyde using two phenoxyl-radical equivalents as oxidants. Finally, the results show that the calixarene platform can be utilized to arrange donor functions to biomimetic binding pockets that allow for the creation of novel types of model compounds.


Assuntos
Calixarenos/química , Galactose Oxidase/química , Oxigênio/química , Aldeídos/química , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Complexos de Coordenação/química , Cobre/química , Técnicas Eletroquímicas/métodos , Ligantes , Modelos Moleculares , Estrutura Molecular , Níquel/química , Oxirredução , Fenóis/química , Ligação Proteica , Zinco/química
19.
Cell Biochem Biophys ; 77(3): 203-211, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31446553

RESUMO

Covalent inhibition targeting noncatalytic residues is rapidly gaining attention in drug discovery. Protein tyrosine phosphatases 1B (PTP1B) is an attractive target for therapeutic interventions in cancer and other diseases. Two binding sites of PTP1B enzyme were identified, catalytic and allosteric. The catalytic site is deep and narrow which protects the active site amino acid residue Cys215 from covalent inhibition, whereas the allosteric site is more hydrophobic and less conserved with Cys121 residue, to which covalent inhibitors can bind. A recent experimental report highlighted that a highly selective inhibitor, 73U, was found to bind covalently in the allosteric region of PTP1B enzyme. Using a robust covalent simulations protocol which was developed in-house, we explore the origin and impact of covalent inhibition upon inhibitor binding to allosteric site. For this, covalently bound and apo enzymes were investigated. Results revealed that allosteric covalent inhibition has ensued in a significant disturbance in the overall network of interaction between Cys121 and other nearby residues, more specifically Tyr124 and His214. The covalent inhibition also exhibited better protein stability as evident from positive correlation between residues in the allosteric site and multiple van der Waal, hydrogen bond and ionic interactions. Surface analysis revealed an increase in the accessible surface area in order to facilitate for the covalent inhibitor to sink in. These findings indicate that exploring allosteric covalent mechanism of PTP1B enzyme offers an opportunity to develop novel PTP1B covalent inhibitors with high potency and selectivity for cancer and other diseases.


Assuntos
Inibidores Enzimáticos/química , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Sítio Alostérico , Domínio Catalítico , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/uso terapêutico , Humanos , Ligações de Hidrogênio , Simulação de Dinâmica Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores
20.
Nucleic Acids Res ; 47(18): 9637-9657, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31410472

RESUMO

Establishing causal relationship between epigenetic marks and gene transcription requires molecular tools, which can precisely modify specific genomic regions. Here, we present a modular and extensible CRISPR/dCas9-based toolbox for epigenetic editing and direct gene regulation. It features a system for expression of orthogonal dCas9 proteins fused to various effector domains and includes a multi-gRNA system for simultaneous targeting dCas9 orthologs to up to six loci. The C- and N-terminal dCas9 fusions with DNMT3A and TET1 catalytic domains were thoroughly characterized. We demonstrated simultaneous use of the DNMT3A-dSpCas9 and TET1-dSaCas9 fusions within the same cells and showed that imposed cytosine hyper- and hypo-methylation altered level of gene transcription if targeted CpG sites were functionally relevant. Dual epigenetic manipulation of the HNF1A and MGAT3 genes, involved in protein N-glycosylation, resulted in change of the glycan phenotype in BG1 cells. Furthermore, simultaneous targeting of the TET1-dSaCas9 and VPR-dSpCas9 fusions to the HNF1A regulatory region revealed strong and persistent synergistic effect on gene transcription, up to 30 days following cell transfection, suggesting involvement of epigenetic mechanisms in maintenance of the reactivated state. Also, modulation of dCas9 expression effectively reduced off-target effects while maintaining the desired effects on target regions.


Assuntos
Sistemas CRISPR-Cas/genética , Epigênese Genética , Edição de Genes/métodos , Transcrição Genética , Aciltransferases/genética , Domínio Catalítico/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Regulação da Expressão Gênica/genética , Genoma/genética , Glicosilação , Fator 1-alfa Nuclear de Hepatócito/genética , Humanos , Oxigenases de Função Mista/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , RNA Guia/genética
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