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1.
Nat Commun ; 12(1): 4848, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381037

RESUMO

There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two "druggable" pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.


Assuntos
Metiltransferases/química , RNA Helicases/química , SARS-CoV-2/química , Proteínas não Estruturais Virais/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Apoenzimas/química , Apoenzimas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Modelos Moleculares , Fosfatos/química , Fosfatos/metabolismo , Conformação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/metabolismo , RNA Viral/química , RNA Viral/metabolismo , SARS-CoV-2/enzimologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo
2.
Proteins ; 89(9): 1216-1225, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33983654

RESUMO

The main protease Mpro , 3CLpro is an important target from coronaviruses. In spite of having 96% sequence identity among Mpros from SARS-CoV-1 and SARS-CoV-2; the inhibitors used to block the activity of SARS-CoV-1 Mpro so far, were found to have differential inhibitory effect on Mpro of SARS-CoV-2. The possible reason could be due to the difference of few amino acids among the peptidases. Since, overall 3-D crystallographic structure of Mpro from SARS-CoV-1 and SARS-CoV-2 is quite similar and mapping a subtle structural variation is seemingly impossible. Hence, we have attempted to study a structural comparison of SARS-CoV-1 and SARS-CoV-2 Mpro in apo and inhibitor bound states using protein structure network (PSN) based approach at contacts level. The comparative PSNs analysis of apo Mpros from SARS-CoV-1 and SARS-CoV-2 uncovers small but significant local changes occurring near the active site region and distributed throughout the structure. Additionally, we have shown how inhibitor binding perturbs the PSG and the communication pathways in Mpros . Moreover, we have also investigated the network connectivity on the quaternary structure of Mpro and identified critical residue pairs for complex formation using three centrality measurement parameters along with the modularity analysis. Taken together, these results on the comparative PSN provide an insight into conformational changes that may be used as an additional guidance towards specific drug development.


Assuntos
Proteases 3C de Coronavírus/química , Vírus da SARS/enzimologia , SARS-CoV-2/enzimologia , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/metabolismo , Sítios de Ligação , Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Modelos Moleculares , Inibidores de Proteases/farmacologia , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos
3.
FEBS J ; 288(19): 5723-5736, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33783128

RESUMO

Several archaea harbor genes that code for fructosyltransferase (FTF) enzymes. These enzymes have not been characterized yet at structure-function level, but are of extreme interest in view of their potential role in the synthesis of novel compounds for food, nutrition, and pharmaceutical applications. In this study, 3D structure of an inulin-type fructan producing enzyme, inulosucrase (InuHj), from the archaeon Halalkalicoccus jeotgali was resolved in its apo form and with bound substrate (sucrose) molecule and first transglycosylation product (1-kestose). This is the first crystal structure of an FTF from halophilic archaea. Its overall five-bladed ß-propeller fold is conserved with previously reported FTFs, but also shows some unique features. The InuHj structure is closer to those of Gram-negative bacteria, with exceptions such as residue E266, which is conserved in FTFs of Gram-positive bacteria and has possible role in fructan polymer synthesis in these bacteria as compared to fructooligosaccharide (FOS) production by FTFs of Gram-negative bacteria. Highly negative electrostatic surface potential of InuHj, due to a large amount of acidic residues, likely contributes to its halophilicity. The complex of InuHj with 1-kestose indicates that the residues D287 in the 4B-4C loop, Y330 in 4D-5A, and D361 in the unique α2 helix may interact with longer FOSs and facilitate the binding of longer FOS chains during synthesis. The outcome of this work will provide targets for future structure-function studies of FTF enzymes, particularly those from archaea.


Assuntos
Apoenzimas/ultraestrutura , Halobacteriaceae/ultraestrutura , Hexosiltransferases/ultraestrutura , Conformação Proteica , Apoenzimas/química , Archaea/enzimologia , Archaea/ultraestrutura , Cristalografia por Raios X , Halobacteriaceae/enzimologia , Hexosiltransferases/química , Dobramento de Proteína , Sacarose/química , Trissacarídeos/química
4.
Biochem Biophys Res Commun ; 553: 85-91, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33765558

RESUMO

Glucose-6-phosphate dehydrogenase is the first enzyme in the pentose phosphate pathway. The reaction catalyzed by the enzyme is considered to be the main source of reducing power for nicotinamide adenine dinucleotide phosphate (NADPH) and is a precursor of 5-carbon sugar used by cells. To uncover the structural features of the enzyme, we determined the crystal structures of glucose-6-phosphate dehydrogenase from Kluyveromyces lactis (KlG6PD) in both the apo form and a binary complex with its substrate glucose-6-phosphate. KlG6PD contains a Rossman-like domain for cofactor NADPH binding; it also presents a typical antiparallel ß sheet at the C-terminal domain with relatively the same pattern as those of other homologous structures. Moreover, our structural and biochemical analyses revealed that Lys153 contributes significantly to substrate G6P recognition. This study may provide insights into the structural variation and catalytic features of the G6PD enzyme.


Assuntos
Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/metabolismo , Kluyveromyces/enzimologia , Sequência de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Glucosefosfato Desidrogenase/genética , Cinética , Modelos Moleculares , Mutagênese , Relação Estrutura-Atividade , Especificidade por Substrato
5.
Biochem J ; 478(4): 943-959, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33565573

RESUMO

Members of the glycoside hydrolase family 4 (GH4) employ an unusual glycosidic bond cleavage mechanism utilizing NAD(H) and a divalent metal ion, under reducing conditions. These enzymes act upon a diverse range of glycosides, and unlike most other GH families, homologs here are known to accommodate both α- and ß-anomeric specificities within the same active site. Here, we report the catalytic properties and the crystal structures of TmAgu4B, an α-d-glucuronidase from the hyperthermophile Thermotoga maritima. The structures in three different states include the apo form, the NADH bound holo form, and the ternary complex with NADH and the reaction product d-glucuronic acid, at 2.15, 1.97 and 1.85 Šresolutions, respectively. These structures reveal the step-wise route of conformational changes required in the active site to achieve the catalytically competent state, and illustrate the direct role of residues that determine the reaction mechanism. Furthermore, a structural transition of a helical region in the active site to a turn geometry resulting in the rearrangement of a unique arginine residue governs the exclusive glucopyranosiduronic acid recognition in TmAgu4B. Mutational studies show that modifications of the glycone binding site geometry lead to catalytic failure and indicate overlapping roles of specific residues in catalysis and substrate recognition. The data highlight hitherto unreported molecular features and associated active site dynamics that determine the structure-function relationships within the unique GH4 family.


Assuntos
Proteínas de Bactérias/química , Apoenzimas/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Catálise , Domínio Catalítico , Cristalografia por Raios X , Ditiotreitol/metabolismo , Ácido Glucurônico/química , Ácido Glucurônico/metabolismo , Glicosídeo Hidrolases/metabolismo , Holoenzimas/química , Cinética , Manganês/metabolismo , Modelos Moleculares , Família Multigênica , Mutagênese Sítio-Dirigida , NAD/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Thermotoga maritima/enzimologia , Thermotoga maritima/genética
6.
Biochem Biophys Res Commun ; 540: 90-94, 2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33450485

RESUMO

MapA is a histidine acid phosphatase (HAP) from Legionella pneumophila that catalyzes the hydroxylation of a phosphoryl group from phosphomonoesters by an active-site histidine. Several structures of HAPs, including MapA, in complex with the inhibitor tartrate have been solved and the substrate binding tunnel identified; however, the substrate recognition mechanism remains unknown. To gain insight into the mechanism of substrate recognition, the crystal structures of apo-MapA and the MapAD281A mutant in complex with 5'-AMP were solved at 2.2 and 2.6 Å resolution, respectively. The structure of the MapAD281A/5'-AMP complex reveals that the 5'-AMP fits fully into the substrate binding tunnel, with the 2'-hydroxyl group of the ribose moiety stabilized by Glu201 and the adenine moiety sandwiched between His205 and Phe237. This is the second structure of a HAP/AMP complex solved with 5'-AMP binding in a unique manner in the active site. The structure presents a new substrate recognition mechanism of HAPs.


Assuntos
Fosfatase Ácida/química , Fosfatase Ácida/metabolismo , Histidina/metabolismo , Legionella pneumophila/enzimologia , Fosfatase Ácida/genética , Adenina/metabolismo , Sequência de Aminoácidos , Apoenzimas/metabolismo , Domínio Catalítico , Legionella pneumophila/genética , Modelos Moleculares , Mutação , Fenilalanina/metabolismo , Ligação Proteica , Ribose/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Tartaratos/metabolismo
7.
Int J Biol Macromol ; 165(Pt B): 2349-2362, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-33098904

RESUMO

NAD synthetase (NadE) catalyzes the last step in NAD biosynthesis, transforming deamido-NAD+ into NAD+ by a two-step reaction with co-substrates ATP and amide donor ammonia. In this study, we report the crystal structure of Staphylococcus aureus NAD synthetase enzyme (saNadE) at 2.3 Å resolution. We used this structure to perform molecular dynamics simulations of apo-enzyme, enzyme-substrate (NadE with ATP and NaAD) and enzyme-intermediate complexes (NadE with NaAD-AMP) to investigate key binding interactions and explore the conformational transitions and flexibility of the binding pocket. Our results show large shift of N-terminal region in substrate bound form which is important for ATP binding. Substrates drive the correlated movement of loop regions surrounding it as well as some regions distal to the active site and stabilize them at complex state. Principal component analysis of atomic projections distinguish feasible trajectories to delineate distinct motions in enzyme-substrate to enzyme-intermediate states. Our results suggest mixed binding involving dominant induced fit and conformational selection. MD simulation extracted ensembles of NadE could potentially be utilized for in silico screening and structure based design of more effective Methicillin Resistant Staphylococcus aureus (MRSA) inhibitors.


Assuntos
Amida Sintases/química , Cristalografia por Raios X , Staphylococcus aureus Resistente à Meticilina/enzimologia , Simulação de Dinâmica Molecular , Apoenzimas/química , Domínio Catalítico , Estabilidade Enzimática , Humanos , Ligação de Hidrogênio , NAD/biossíntese , Análise de Componente Principal , Conformação Proteica , Subunidades Proteicas/química , Especificidade por Substrato
8.
Int J Biol Macromol ; 165(Pt A): 205-213, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32991904

RESUMO

The antioxidant and antibacterial activities of camel and bovine α-lactalbumin (α-La) in both calcium-loaded (holo) and calcium-depleted (apo) forms were investigated and compared. Antioxidant assay showed that camel and bovine α-La exhibited significant Ferric-reducing antioxidant power (FRAP), ferrous iron-chelating activity (FCA) and antiradical activities especially in their apo form. Camel apo α-La also exhibited attractive antibacterial activities against Gram-negative bacteria (Pseudomonas aeruginosa) and against fungal pathogens species (Penicillium bilaiae, Aspergillus tamari and Aspergillus sclerotiorum). Likewise, emulsifying properties (emulsification ability (EAI) and stability (ESI) indexes) and the surface characteristics (surface hydrophobicity, ζ-potential and interfacial tension) of the α-La were assessed. Maximum EAI were found at pH 7.0, with higher EAI values for the camel apo α-La (EAI ~19.5 m2/g). This behavior was explained by its relative high surface hydrophobicity and its greater efficiency to reduce the surface tension at the oil-water interface. Furthermore, emulsions were found to be more stable at pH 7.0 compared to pH 5.0 (ESI ~50%) due to the higher electrostatic repulsive forces between oil droplets at pH 7.0 in consistence with the ζ-potential results. This study concluded that the camel apo α-La has antibacterial, antioxidant, and emulsifying properties in agricultural and food industries.


Assuntos
Antibacterianos/química , Antioxidantes/química , Lactalbumina/química , Animais , Antibacterianos/farmacologia , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Apoenzimas/química , Apoenzimas/isolamento & purificação , Aspergillus/efeitos dos fármacos , Camelus , Bovinos , Emulsões/química , Emulsões/farmacologia , Holoenzimas/química , Holoenzimas/isolamento & purificação , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Lactalbumina/isolamento & purificação , Lactalbumina/farmacologia , Penicillium/efeitos dos fármacos
9.
FEBS J ; 287(24): 5375-5393, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32255258

RESUMO

Williams-Beuren syndrome, characterized by numerous physiological and mental problems, is caused by the heterozygous deletion of chromosome region 7q11.23, which results in the disappearance of 26 protein-coding genes. Protein WBSCR27 is a product of one of these genes whose biological function has not yet been established and for which structural information has been absent until now. Using NMR, we investigated the structural and functional properties of murine WBSCR27. For protein in the apo form and in a complex with S-(5'-adenosyl)-l-homocysteine (SAH), a complete NMR resonance assignment has been obtained and the secondary structure has been determined. This information allows us to attribute WBSCR27 to Class I methyltransferases. The interaction of WBSCR27 with the cofactor S-(5'-adenosyl)-l-methionine (SAM) and its metabolic products - SAH, 5'-deoxy-5'-methylthioadenosine (MTA) and 5'-deoxyadenosine (5'dAdo) - was studied by NMR and isothermal titration calorimetry. SAH binds WBSCR27 much tighter than SAM, leaving open the question of cofactor turnover in the methylation reaction. One possible answer to this question is the presence of weak but detectable nucleosidase activity for WBSCR27. We found that the enzyme catalyses the cleavage of the adenine moiety from SAH, MTA and 5'dAdo, similar to the action of bacterial SAH/MTA nucleosidases. We also found that the binding of SAM or SAH causes a significant change in the structure of WBSCR27 and in the conformational mobility of the protein fragments, which can be attributed to the substrate recognition site. This indicates that the binding of the cofactor modulates the folding of the substrate-recognizing region of the enzyme.


Assuntos
Desoxiadenosinas/metabolismo , Metiltransferases/química , Metiltransferases/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Tionucleosídeos/metabolismo , Animais , Apoenzimas , Camundongos , Conformação Proteica
10.
Nature ; 579(7800): 615-619, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32214249

RESUMO

Arenaviruses can cause severe haemorrhagic fever and neurological diseases in humans and other animals, exemplified by Lassa mammarenavirus, Machupo mammarenavirus and lymphocytic choriomeningitis virus, posing great threats to public health1-4. These viruses encode a large multi-domain RNA-dependent RNA polymerase for transcription and replication of the viral genome5. Viral polymerases are one of the leading antiviral therapeutic targets. However, the structure of arenavirus polymerase is not yet known. Here we report the near-atomic resolution structures of Lassa and Machupo virus polymerases in both apo and promoter-bound forms. These structures display a similar overall architecture to influenza virus and bunyavirus polymerases but possess unique local features, including an arenavirus-specific insertion domain that regulates the polymerase activity. Notably, the ordered active site of arenavirus polymerase is inherently switched on, without the requirement for allosteric activation by 5'-viral RNA, which is a necessity for both influenza virus and bunyavirus polymerases6,7. Moreover, dimerization could facilitate the polymerase activity. These findings advance our understanding of the mechanism of arenavirus replication and provide an important basis for developing antiviral therapeutics.


Assuntos
Arenavirus do Novo Mundo/enzimologia , Microscopia Crioeletrônica , Vírus Lassa/enzimologia , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/ultraestrutura , Replicação Viral , Apoenzimas/química , Apoenzimas/metabolismo , Apoenzimas/ultraestrutura , Arenavirus do Novo Mundo/ultraestrutura , Domínio Catalítico , Vírus Lassa/ultraestrutura , Vírus da Coriomeningite Linfocítica/enzimologia , Vírus da Coriomeningite Linfocítica/ultraestrutura , Modelos Moleculares , Regiões Promotoras Genéticas/genética , RNA Polimerase Dependente de RNA/metabolismo
11.
Protein Eng Des Sel ; 32(2): 77-85, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31832682

RESUMO

Intracellular aggregates of superoxide dismutase 1 (SOD1) are associated with amyotrophic lateral sclerosis. In vivo, aggregation occurs in a complex and dense molecular environment with chemically heterogeneous surfaces. To investigate how SOD1 fibril formation is affected by surfaces, we used an in vitro model system enabling us to vary the molecular features of both SOD1 and the surfaces, as well as the surface area. We compared fibril formation in hydrophilic and hydrophobic sample wells, as a function of denaturant concentration and extraneous hydrophobic surface area. In the presence of hydrophobic surfaces, SOD1 unfolding promotes fibril nucleation. By contrast, in the presence of hydrophilic surfaces, increasing denaturant concentration retards the onset of fibril formation. We conclude that the mechanism of fibril formation depends on the surrounding surfaces and that the nucleating species might correspond to different conformational states of SOD1 depending on the nature of these surfaces.


Assuntos
Amiloide/química , Biocatálise , Interações Hidrofóbicas e Hidrofílicas , Agregados Proteicos , Desdobramento de Proteína , Superóxido Dismutase-1/química , Superóxido Dismutase-1/metabolismo , Adsorção , Apoenzimas/química , Apoenzimas/metabolismo , Dissulfetos/química , Propriedades de Superfície
12.
Nature ; 575(7783): 540-544, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31723264

RESUMO

Transposons have had a pivotal role in genome evolution1 and are believed to be the evolutionary progenitors of the RAG1-RAG2 recombinase2, an essential component of the adaptive immune system in jawed vertebrates3. Here we report one crystal structure and five cryo-electron microscopy structures of Transib4,5, a RAG1-like transposase from Helicoverpa zea, that capture the entire transposition process from the apo enzyme to the terminal strand transfer complex with transposon ends covalently joined to target DNA, at resolutions of 3.0-4.6 Å. These structures reveal a butterfly-shaped complex that undergoes two cycles of marked conformational changes in which the 'wings' of the transposase unfurl to bind substrate DNA, close to execute cleavage, open to release the flanking DNA and close again to capture and attack target DNA. Transib possesses unique structural elements that compensate for the absence of a RAG2 partner, including a loop that interacts with the transposition target site and an accordion-like C-terminal tail that elongates and contracts to help to control the opening and closing of the enzyme and assembly of the active site. Our findings reveal the detailed reaction pathway of a eukaryotic cut-and-paste transposase and illuminate some of the earliest steps in the evolution of the RAG recombinase.


Assuntos
Biocatálise , Proteínas de Homeodomínio , Mariposas/enzimologia , Transposases/química , Transposases/metabolismo , Sequência de Aminoácidos , Animais , Apoenzimas/química , Apoenzimas/metabolismo , Apoenzimas/ultraestrutura , Sequência de Bases , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA/química , DNA/genética , DNA/metabolismo , Clivagem do DNA , Proteínas de Ligação a DNA , Evolução Molecular , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/ultraestrutura , Modelos Moleculares , Mariposas/ultraestrutura , Domínios Proteicos , Transposases/ultraestrutura
13.
Int J Biol Macromol ; 136: 676-685, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31207333

RESUMO

The M. tuberculosis GmhB protein converts the d-glycero-α-d-manno-heptose 1,7-bisphosphate (GMB) intermediate into d-glycero-α-d-manno-heptose 1-phosphate by removing the phosphate group at the C-7 position. To understand the structure and substrate binding mechanism, the MtbGmhB was purified which elutes as monomer on gel filtration column. The small angle x-ray scattering analysis shows that MtbGmhB forms fully folded monomer with shape profile similar to its modeled structure. The circular dichroism analysis shows 38% α-helix, 15% ß-sheets and 47% random coil structures in MtbGmhB, similar to haloalkanoic acid dehalogenase (HAD) phosphohydrolase enzymes. The modeled MtbGmhB structure shows the catalytic site, which forms a concave, semicircular surface using the three loops around GMB substrate binding site. Dynamic simulation analysis on (i) Apo (ii) GMB bound (iii) GMB + Mg2+ bound (iv) Zn2+ +GMB + Mg2+ bound MtbGmhB structures show that Zn2+ as well as Mg2+ ions stabilize the loop conformation and trigger the changes in GMB substrate binding to active site of MtbGmhB. Upon demetallization, the large conformational changes occurred in ions binding loops, and leads to difference in GMB substrate binding to MtbGmhB. Our study provides information about structure and substrate binding of MtbGmhB, which may contribute in therapeutic development against M. tuberculosis.


Assuntos
Guanosina Difosfato/biossíntese , Heptoses/biossíntese , Mycobacterium tuberculosis/enzimologia , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X , Sequência de Aminoácidos , Apoenzimas/química , Apoenzimas/metabolismo , Domínio Catalítico , Magnésio/metabolismo , Simulação de Acoplamento Molecular , Zinco/metabolismo
14.
Biophys J ; 116(10): 1823-1835, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31003762

RESUMO

A critical step in injury-induced initiation of blood coagulation is the formation of the complex between the trypsin-like protease coagulation factor VIIa (FVIIa) and its cofactor tissue factor (TF), which converts FVIIa from an intrinsically poor enzyme to an active protease capable of activating zymogens of downstream coagulation proteases. Unlike its constitutively active ancestor trypsin, FVIIa is allosterically activated (by TF). Here, ensemble refinement of crystallographic structures, which uses multiple copies of the entire structure as a means of representing structural flexibility, is applied to explore the impacts of inhibitor binding to trypsin and FVIIa, as well as cofactor binding to FVIIa. To assess the conformational flexibility and its role in allosteric pathways in these proteases, main-chain hydrogen bond networks are analyzed by calculating the hydrogen-bond propensity. Mapping pairwise propensity differences between relevant structures shows that binding of the inhibitor benzamidine to trypsin has a minor influence on the protease flexibility. For FVIIa, in contrast, the protease domain is "locked" into the catalytically competent trypsin-like configuration upon benzamidine binding as indicated by the stabilization of key structural features: the nonprime binding cleft and the oxyanion hole are stabilized, and the effect propagates from the active site region to the calcium-binding site and to the vicinity of the disulphide bridge connecting with the light chain. TF binding to FVIIa furthermore results in stabilization of the 170 loop, which in turn propagates an allosteric signal from the TF-binding region to the active site. Analyses of disulphide bridge energy and flexibility reflect the striking stability difference between the unregulated enzyme and the allosterically activated form after inhibitor or cofactor binding. The ensemble refinement analyses show directly, for the first time to our knowledge, whole-domain structural footprints of TF-induced allosteric networks present in x-ray crystallographic structures of FVIIa, which previously only have been hypothesized or indirectly inferred.


Assuntos
Fator VIIa/química , Fator VIIa/metabolismo , Regulação Alostérica , Apoenzimas/química , Apoenzimas/metabolismo , Benzamidinas/farmacologia , Cristalografia por Raios X , Dissulfetos/química , Ativação Enzimática/efeitos dos fármacos , Modelos Moleculares , Domínios Proteicos , Dobramento de Proteína , Tripsina/química , Tripsina/metabolismo , Tripsinogênio/metabolismo
15.
Int J Biol Macromol ; 132: 994-1000, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30953724

RESUMO

The structure and folding/unfolding kinetics of Cyathus bulleri laccase 1 (Lcc1), expressed in Pichia pastoris, were analyzed by spectroscopic methods to understand the role of central metal ion. Far UV CD structure analysis revealed major ß-sheet and minor α helical segments present in the Lcc1. A significant change in the spectrum of Lcc1, indicative of unfolding of secondary structures, was observed with increasing concentrations of guanidinium chloride (GdnHCl) during Trp fluorescence, absorption and CD measurements. A similar trend was also noticed for enzyme activity with respect to GdnHCl concentrations. To establish the role of copper ion in the catalytic activity of laccase, a complete removal of copper was carried out and addition of copper was found to restore the structure and activity during the refolding process. The apo form was also reconstituted by addition of zinc ion which restored nearly 84% of enzyme activity, indicating non-essential role of copper in maintaining conformation and activity. Structural studies and inductively coupled plasma mass spectrometry data supported these observations.


Assuntos
Cobre , Cyathus/enzimologia , Guanidina/farmacologia , Lacase/química , Lacase/metabolismo , Desnaturação Proteica/efeitos dos fármacos , Apoenzimas/química , Apoenzimas/metabolismo , Biocatálise , Relação Dose-Resposta a Droga , Redobramento de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína , Termodinâmica , Zinco
16.
Int J Mol Sci ; 20(5)2019 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-30836629

RESUMO

Human triokinase/flavin mononucleotide (FMN) cyclase (hTKFC) catalyzes the adenosine triphosphate (ATP)-dependent phosphorylation of D-glyceraldehyde and dihydroxyacetone (DHA), and the cyclizing splitting of flavin adenine dinucleotide (FAD). hTKFC structural models are dimers of identical subunits, each with two domains, K and L, with an L2-K1-K2-L1 arrangement. Two active sites lie between L2-K1 and K2-L1, where triose binds K and ATP binds L, although the resulting ATP-to-triose distance is too large (≈14 Å) for phosphoryl transfer. A 75-ns trajectory of molecular dynamics shows considerable, but transient, ATP-to-DHA approximations in the L2-K1 site (4.83 Å or 4.16 Å). To confirm the trend towards site closure, and its relationship to kinase activity, apo-hTKFC, hTKFC:2DHA:2ATP and hTKFC:2FAD models were submitted to normal mode analysis. The trajectory of hTKFC:2DHA:2ATP was extended up to 160 ns, and 120-ns trajectories of apo-hTKFC and hTKFC:2FAD were simulated. The three systems were comparatively analyzed for equal lengths (120 ns) following the principles of essential dynamics, and by estimating site closure by distance measurements. The full trajectory of hTKFC:2DHA:2ATP was searched for in-line orientations and short distances of DHA hydroxymethyl oxygens to ATP γ-phosphorus. Full site closure was reached only in hTKFC:2DHA:2ATP, where conformations compatible with an associative phosphoryl transfer occurred in L2-K1 for significant trajectory time fractions.


Assuntos
Apoenzimas/genética , Simulação de Dinâmica Molecular , Fósforo-Oxigênio Liases/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Trifosfato de Adenosina/química , Apoenzimas/química , Sítios de Ligação , Catálise , Domínio Catalítico/genética , Di-Hidroxiacetona/química , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/genética , Flavina-Adenina Dinucleotídeo/química , Gliceraldeído/química , Humanos , Fósforo-Oxigênio Liases/genética , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Especificidade por Substrato
17.
Int J Biol Macromol ; 129: 588-600, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30703421

RESUMO

Salicylate hydroxylase (NahG) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of salicylate into catechol in the naphthalene degradation pathway in Pseudomonas putida G7. We explored the mechanism of action of this enzyme in detail using a combination of structural and biophysical methods. NahG shares many structural and mechanistic features with other versatile flavin-dependent monooxygenases, with potential biocatalytic applications. The crystal structure at 2.0 Šresolution for the apo form of NahG adds a new snapshot preceding the FAD binding in flavin-dependent monooxygenases. The kcat/Km for the salicylate reaction catalyzed by the holo form is >105 M-1 s-1 at pH 8.5 and 25 °C. Hammett plots for Km and kcat using substituted salicylates indicate change in rate-limiting step. Electron-donating groups favor the hydroxylation of salicylate by a peroxyflavin to yield a Wheland-like intermediate, whereas the decarboxylation of this intermediate is faster for electron-withdrawing groups. The mechanism is supported by structural data and kinetic studies at different pHs. The salicylate carboxyl group lies near a hydrophobic region that aids decarboxylation. A conserved histidine residue is proposed to assist the reaction by general base/general acid catalysis.


Assuntos
Biocatálise , Catecóis/metabolismo , Dinitrocresóis/metabolismo , Oxigenases de Função Mista/metabolismo , Ácido Salicílico/metabolismo , Apoenzimas/química , Apoenzimas/metabolismo , Domínio Catalítico , Cinética , Oxigenases de Função Mista/química , Modelos Moleculares , Pseudomonas putida/enzimologia , Termodinâmica
18.
Methods Mol Biol ; 1866: 107-131, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30725412

RESUMO

The elevated requirement for methionine (MET) of cancer cells is termed MET dependence. To selectively target the MET dependence of tumors for treatment on a large-scale preclinical and clinical basis, the L-methionine α-deamino-γ-mercaptomethane-lyase (EC 4.4.1.11) (methioninase, [METase]) gene from Pseudomonas putida has been cloned in Escherichia coli using the polymerase chain reaction (PCR). Purification using two DEAE Sepharose FF ion-exchange column and one ActiClean Etox endotoxin-affinity chromatography column has been established. Plasmid pMGLTrc03, which has a trc promoter and a spacing of 12 nucleotides between the Shine-Dalgarno sequence and the ATG translation initiation codon, was selected as the most suitable plasmid. The recombinant bacteria produced rMETase at 43% of the total proteins in soluble fraction by simple batch fermentation using a 500 L fermentor. Crystals were directly obtained from crude enzyme with 87% yield by a crystallization in the presence of 9.0% polyethylene glycol 6000, 3.6% ammonium sulfate, and 0.18 M sodium chloride using a 100 L crystallizer. After recrystallization, the enzyme was purified by anion-exchange column chromatography to remove endotoxins and by gel filtration for polishing. Purified rMETase is stable to lyophilization. In order to prevent immunological reactions which might be produced by multiple dosing of rMETase and to prolong the serum half-life of rMETase, the N-hydroxysuccinimidyl ester of methoxypolyethylene glycol propionic acid (M-SPA-PEG 5000) has been coupled to rMETase. The PEGylated molecules (PEG-rMETase) were purified from unreacted PEG with Amicon 30 K centriprep concentrators or by Sephacryl S-300 HR gel-filtration chromatography. Unreacted rMETase was removed by DEAE Sepharose FF anion-exchange chromatography. The resulting PEG-rMETase subunit, produced from a PEG/rMETase ratio of 30/1 in the synthetic reaction, had a molecular mass of approximately 53 kda determined by matrix-assisted laser desorption/ionization mass spectrometry, indicating the conjugation of two PEG molecules per subunit of rMETase and eight per tetramer. PEG-rMETase molecules obtained from reacting ratios of PEG/rMETase of 30/1 had an enzyme activity of 70% of unmodified rMETase. PEGylation of rMETase increased the serum half-life of the enzyme in rats to approximately 160 min compared to 80 min for unmodified rMETase. PEG-rMETase could deplete serum MET levels to less than 0.1 µM for approximately 8 h compared to 2 h for rMETase in rats. A significant prolongation of in vivo activity and effective MET depletion by the PEG-rMETase were achieved by the simultaneous administration of pyridoxal 5'-phosphate. rMETase was also conjugated with methoxypolyethylene glycol succinimidyl glutarate 5000 (MEGC-PEG). Miniosmotic pumps containing various concentrations of PLP were implanted in BALB-C mice. PLP-infused mice were then injected with a single dose of 4000 or 8000 units/kg PEG-rMETase. Mice infused with 5, 50, 100, 200, and 500 mg/mL PLP-containing miniosmotic pumps increased plasma PLP to 7, 24, 34, 60, and 95 µM, respectively, from the PLP baseline of 0.3 µM. PLP increased the half-life of MEGC-PEG-rMETase holoenzyme in a dose-dependent manner. The extended time of MET depletion by MEGC-PEG-rMETase was due to the maintenance of active MEGC-PEG-rMETase holoenzyme by infused PLP.


Assuntos
Liases de Carbono-Enxofre/uso terapêutico , Neoplasias/tratamento farmacológico , Proteínas Recombinantes/uso terapêutico , Animais , Apoenzimas/metabolismo , Liases de Carbono-Enxofre/sangue , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/isolamento & purificação , Cristalização , Escherichia coli/metabolismo , Fermentação , Camundongos Endogâmicos BALB C , Polietilenoglicóis/química , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Fosfato de Piridoxal/administração & dosagem , Fosfato de Piridoxal/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
19.
J Mol Model ; 24(12): 347, 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30498917

RESUMO

Low-temperature methane oxidation is one of the greatest challenges in energy research. Although methane monooxygenase (MMO) does this catalysis naturally, how to use this biocatalyst in a fuel cell environment where the electrons generated during the oxidation process is harvested and used for energy generation has not yet been investigated. A key requirement to use this enzyme in a fuel cell is wiring of the active site of the enzyme directly to the supporting electrode. In soluble MMO (sMMO), two cofactors, i.e., nicotinamide adenine di-nucleotide (NAD+) and flavin adenine dinucleotide (FAD) provide opportunities for direct attachment of the enzyme system to a supporting electrode. However, once modified to be compatible with a supporting metal electrode via FeS functionalization, how the two cofactors respond to complex binding phenomena is not yet understood. Using docking and molecular dynamic simulations, modified cofactors interactions with sMMO-reductase (sMMOR) were studied. Studies revealed that FAD modification with FeS did not interfere with binding phenomena. In fact, FeS introduction significantly improved the binding affinity of FAD and NAD+ on sMMOR. The simulations revealed a clear thermodynamically more favorable electron transport path for the enzyme system. This system can be used as a fuel cell and we can use FeS-modified-FAD as the anchoring molecule as opposed to using NAD+. The overall analysis suggests the strong possibility of building a fuel cell that could catalyze methane oxidation using sMMO as the anode biocatalyst.


Assuntos
Apoenzimas/química , Proteínas de Bactérias/química , Coenzimas/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Oxigenases/química , Apoenzimas/metabolismo , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Coenzimas/metabolismo , Biologia Computacional/métodos , Transporte de Elétrons , Metano/metabolismo , Methylococcus capsulatus/enzimologia , Oxigenases/metabolismo , Ligação Proteica , Domínios Proteicos , Engenharia de Proteínas/métodos , Reprodutibilidade dos Testes , Especificidade por Substrato
20.
Acta Crystallogr F Struct Biol Commun ; 74(Pt 10): 610-616, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30279311

RESUMO

Three high-resolution X-ray crystal structures of malate dehydrogenase (MDH; EC 1.1.1.37) from the methylotroph Methylobacterium extorquens AM1 are presented. By comparing the structures of apo MDH, a binary complex of MDH and NAD+, and a ternary complex of MDH and oxaloacetate with ADP-ribose occupying the pyridine nucleotide-binding site, conformational changes associated with the formation of the catalytic complex were characterized. While the substrate-binding site is accessible in the enzyme resting state or NAD+-bound forms, the substrate-bound form exhibits a closed conformation. This conformational change involves the transition of an α-helix to a 310-helix, which causes the adjacent loop to close the active site following coenzyme and substrate binding. In the ternary complex, His284 forms a hydrogen bond to the C2 carbonyl of oxaloacetate, placing it in a position to donate a proton in the formation of (2S)-malate.


Assuntos
Adenosina Difosfato Ribose/química , Proteínas de Bactérias/química , Malato Desidrogenase/química , Malatos/química , Methylobacterium extorquens/química , NAD/química , Ácido Oxaloacético/química , Adenosina Difosfato Ribose/metabolismo , Sequência de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ligação de Hidrogênio , Cinética , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Malatos/metabolismo , Methylobacterium extorquens/enzimologia , Modelos Moleculares , NAD/metabolismo , Ácido Oxaloacético/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Prótons , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
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