RESUMEN
Phosphopantetheine adenylyltransferase (EC. 2.7.7.3, PPAT) catalyzes the penultimate step of the multistep reaction in the coenzyme A (CoA) biosynthesis pathway. In this step, an adenylyl group from adenosine triphosphate (ATP) is transferred to 4'-phosphopantetheine (PNS) yielding 3'-dephospho-coenzyme A (dpCoA) and pyrophosphate (PPi). PPAT from strain C3 of Klebsiella pneumoniae (KpPPAT) was cloned, expressed and purified. It was crystallized using 0.1 M HEPES buffer and PEG10000 at pH 7.5. The crystals belonged to tetragonal space group P41212 with cell dimensions of a = b = 72.82 Å and c = 200.37 Å. The structure was determined using the molecular replacement method and refined to values of 0.208 and 0.255 for Rcryst and Rfree factors, respectively. The structure determination showed the presence of three crystallographically independent molecules A, B and C in the asymmetric unit. The molecules A and B are observed in the form of a dimer in the asymmetric unit while molecule C belongs to the second dimer whose partner is related by crystallographic twofold symmetry. The polypeptide chain of KpPPAT folds into a ß/α structure. The conformations of the side chains of several residues in the substrate binding site in KpPPAT are significantly different from those reported in other PPATs. As a result, the modes of binding of substrates, phosphopantetheine (PNS) and adenosine triphosphate (ATP) differ considerably. The binding studies using fluorescence spectroscopy indicated a KD value of 3.45 × 10-4 M for ATP which is significantly lower than the corresponding values reported for PPAT from other species.
Asunto(s)
Adenosina Trifosfato , Klebsiella pneumoniae , Nucleotidiltransferasas , Klebsiella pneumoniae/metabolismo , Cristalografía por Rayos X , Coenzima A/química , Coenzima A/metabolismoRESUMEN
We draw the attention of readers and governments to the death rate from coronavirus disease 2019 in Japan, continuing as a fraction of that experienced by many other developed nations. We think this is due to the activity of the powerful, protective lactoperoxidase system (LPO) which prevents serious airborne infections. The LPO system requires iodine, which is liberally provided by the typical Japanese diet but lacking in many others. One might consider the Japanese experience an incredibly large, open-label study exhibiting the preventative power of a high-iodine diet. We predict this favourable trend will continue for Japan because deadly variants of the severe, acute respiratory syndrome coronavirus 2 will be with us, forever.
Asunto(s)
COVID-19 , Yodo , Humanos , Japón/epidemiología , Lactoperoxidasa , SARS-CoV-2RESUMEN
The mammalian lactoperoxidase system, consisting of lactoperoxidase and the H2 O2 -producing enzyme duox, is our first line of defence against airborne microbes. This system catalyses the production of hypoiodite and hypoiodous acid in the presence of sufficient iodine. These products are highly efficient at destroying the H1N1 virus and the respiratory syncytial virus (RSV). Japan has not been affected as much as other nations during the COVID-19 pandemic (death rate about 10% of the United States), and we think this is due to a diet high in iodine. With this in mind, we suggest four actions to prevent SARS-CoV-2 infections. First, health professionals should study the preventative effect of increasing iodine in the diets of the aged, institutionalized, diabetics andsmokers. Second, the recommended daily intake (RDI) for iodine should be significantly increased, to at least double, the current RDI. Governments should encourage the use and distribution of cheap iodized salts, kelp and seaweed. Third, more research should be done around the physiology and the protective effects of the lactoperoxidase system. Finally, the degradation products of the SARS-CoV-2 viral particle by hypoiodite and hypoiodous acid should be characterized; portions of the damaged particle are likely to elicit stronger immunity and better vaccines.
Asunto(s)
COVID-19/dietoterapia , COVID-19/prevención & control , Dietoterapia/métodos , Yodo/administración & dosificación , SARS-CoV-2/efectos de los fármacos , COVID-19/epidemiología , Dieta , Humanos , Inmunomodulación/inmunología , Compuestos de Yodo/metabolismo , Japón/epidemiología , Lactoperoxidasa/metabolismoRESUMEN
Acinetobacter baumannii is a multidrug-resistant, opportunistic, nosocomial pathogen for which a new line of treatments is desperately needed. We have targeted the enzyme of the first step of the histidine biosynthesis pathway, viz., ATP-phosphoribosyltransferase (ATP-PRT). The three-dimensional structure of ATP-PRT was predicted on the template of the known three-dimensional structure of ATP-PRT from Psychrobacter arcticus (PaATPPRT) using a homology modeling approach. High-throughput virtual screening (HTVS) of the antibacterial library of Life Chemicals Inc., Ontario, Canada was carried out followed by molecular dynamics simulations of the top hit compounds. In silico results were then biochemically validated using surface plasmon resonance spectroscopy. We found that two compounds, namely, F0843-0019 and F0608-0626, were binding with micromolar affinities to the ATP-phosphoribosyltransferase from Acinetobacter baumannii (AbATPPRT). Both of these compounds were binding in the same way as AMP in PaATPPRT, and the important residues of the active site, viz., Val4, Ser72, Thr76, Tyr77, Glu95, Lys134, Val136, and Tyr156, were also interacting via hydrogen bonds. The calculated binding energies of these compounds were -10.5 kcal/mol and -11.1 kcal/mol, respectively. These two compounds can be used as the potential lead molecules for designing antibacterial compounds in the future, and this information will help in drug discovery programs against Acinetobacter worldwide.
Asunto(s)
Acinetobacter baumannii , Acinetobacter baumannii/metabolismo , Adenosina Trifosfato/metabolismo , Antibacterianos/química , Histidina , Simulación del Acoplamiento MolecularRESUMEN
Lactoperoxidase (LPO) is a glycosylated antimicrobial protein present in milk with a molecular mass of 78 kDa. LPO is included in many biological processes and is well-known to have biocidal actions, acting as an active antibiotic and antiviral agent. The wide spectrum biocidal activity of LPO is mediated via a definite inhibitory system named lactoperoxidase system which plays a potent role in the innate immune response. With the current advancement in nanotechnology, nanoformulations can be developed for stabilizing and potentiating the activity of LPO for several applications. In the research described in this Research Communication, fresh LPO purified from bovine mammary gland secretions was used for nanoparticle synthesis using a simple thermal process at different pH and temperatures. The round-shaped nanoparticles (average size 229 nm) were successfully synthesized at pH 7.0 and a temperature of 75°C. These nanoparticles were tested against four different bacterial species namely S. flexineri, P. aeruginosa, S. aureus, and E. coli. The prepared nanoparticles exhibited strong inhibition of the growth against all four bacterial species as stated by their MIC and ZOI values. These results may help in increasing the efficiency of lactoperoxidase system and will assist in identifying novel avenues to enhance the stability and antimicrobial function of LPO in drug discovery and industrial processes.
Asunto(s)
Antiinfecciosos , Lactoperoxidasa , Animales , Bovinos , Lactoperoxidasa/química , Escherichia coli , Staphylococcus aureus , Leche/química , Antiinfecciosos/farmacologíaRESUMEN
Lactoperoxidase, a heme-containing glycoprotein, catalyzes the oxidation of thiocyanate by hydrogen peroxide into hypothiocyanite which acts as an antibacterial agent. The prosthetic heme moiety is attached to the protein through two ester linkages via Glu258 and Asp108. In lactoperoxidase, the substrate-binding site is formed on the distal heme side. To study the effect of physiologically important potassium ion on the structure and function of lactoperoxidase, the fresh protein samples were isolated from yak (Bos grunniens) colostrum and purified to homogeneity. The biochemical studies with potassium fluoride showed a significant reduction in the catalytic activity. Lactoperoxidase was crystallized using 200 mM ammonium nitrate and 20% PEG-3350 at pH 6.0. The crystals of LPO were soaked in the solution of potassium fluoride and used for the X-ray intensity data collection. Structure determination at 2.20 Šresolution revealed the presence of a potassium ion in the distal heme cavity. Structure determination further revealed that the propionic chain attached to pyrrole ring C of the heme moiety, was disordered into two components each having an occupancy of 0.5. One component occupied a position similar to the normally observed position of propionic chain while the second component was found in the distal heme cavity. The potassium ion in the distal heme cavity formed five coordinate bonds with two oxygen atoms of propionic moiety, Nε2 atom of His109 and two oxygen atoms of water molecules. The presence of potassium ion in the distal heme cavity hampered the catalytic activity of lactoperoxidase.
Asunto(s)
Lactoperoxidasa/metabolismo , Potasio/metabolismo , Animales , Sitios de Unión , Biocatálisis , Calcio/química , Calcio/metabolismo , Bovinos , Calostro/enzimología , Cristalografía por Rayos X , Hemo/química , Hemo/metabolismo , Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/metabolismo , Lactoperoxidasa/química , Potasio/química , Unión ProteicaRESUMEN
The accumulation of massive data in the plethora of Cheminformatics databases has made the role of big data and artificial intelligence (AI) indispensable in drug design. This has necessitated the development of newer algorithms and architectures to mine these databases and fulfil the specific needs of various drug discovery processes such as virtual drug screening, de novo molecule design and discovery in this big data era. The development of deep learning neural networks and their variants with the corresponding increase in chemical data has resulted in a paradigm shift in information mining pertaining to the chemical space. The present review summarizes the role of big data and AI techniques currently being implemented to satisfy the ever-increasing research demands in drug discovery pipelines.
Asunto(s)
Inteligencia Artificial , Macrodatos , Descubrimiento de Drogas/métodos , Algoritmos , Bases de Datos Factuales , Aprendizaje Profundo , Diseño de Fármacos , Aprendizaje Automático , Reproducibilidad de los Resultados , Flujo de TrabajoRESUMEN
Ribosome inactivating protein (RIP) catalyzes the cleavage of glycosidic bond formed between adenine and ribose sugar of ribosomal RNA to inactivate ribosomes. Previous structural studies have shown that RNA bases, adenine, guanine, and cytosine tend to bind to RIP in the substrate binding site. However, the mode of binding of uracil with RIP was not yet known. Here, we report crystal structures of two complexes of type 1 RIP from Momordica balsamina (MbRIP1) with base, uracil and nucleoside, uridine. The binding studies of MbRIP1 with uracil and uridine as estimated using fluorescence spectroscopy showed that the equilibrium dissociation constants (KD ) were 1.2 × 10-6 M and 1.4 × 10-7 M respectively. The corresponding values obtained using surface plasmon resonance (SPR) were found to be 1.4 × 10-6 M and 1.1 × 10-7 M, respectively. Structures of the complexes of MbRIP1 with uracil (Structure-1) and uridine (Structure-2) were determined at 1.70 and 1.98 Å resolutions respectively. Structure-1 showed that uracil bound to MbRIP1 at the substrate binding site but its mode of binding was significantly different from those of adenine, guanine and cytosine. However, the mode of binding of uridine was found to be similar to those of cytidine. As a result of binding of uracil to MbRIP1 at the substrate binding site, three water molecules were expelled while eight water molecules were expelled when uridine bound to MbRIP1.
Asunto(s)
Momordica/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1/metabolismo , Uracilo/química , Uridina/química , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacología , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/farmacología , Unión Proteica , Conformación Proteica , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1/química , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Ribosomas/química , Ribosomas/metabolismo , Resonancia por Plasmón de Superficie , Uracilo/metabolismo , Uracilo/farmacología , Uridina/metabolismo , Uridina/farmacologíaRESUMEN
Peptidyl-tRNA hydrolase (Pth) catalyzes the breakdown of peptidyl-tRNA into peptide and tRNA components. Pth from Acinetobacter baumannii (AbPth) was cloned, expressed, purified and crystallized in a native unbound (AbPth-N) state and in a bound state with the phosphate ion and cytosine arabinoside (cytarabine) (AbPth-C). Structures of AbPth-N and AbPth-C were determined at 1.36 and 1.10â Å resolutions, respectively. The structure of AbPth-N showed that the active site is filled with water molecules. In the structure of AbPth-C, a phosphate ion is present in the active site, while cytarabine is bound in a cleft which is located away from the catalytic site. The cytarabine-binding site is formed with residues: Gln19, Trp27, Glu30, Gln31, Lys152, Gln158 and Asp162. In the structure of AbPth-N, the side chains of two active-site residues, Asn70 and Asn116, were observed in two conformations. Upon binding of the phosphate ion in the active site, the side chains of both residues were ordered to single conformations. Since Trp27 is present at the cytarabine-binding site, the fluorescence studies were carried out which gave a dissociation constant (KD) of 3.3 ± 0.8 × 10-7â M for cytarabine. The binding studies using surface plasmon resonance gave a KD value of 3.7 ± 0.7 × 10-7â M. The bacterial inhibition studies using the agar diffusion method and the biofilm inhibition assay established the strong antimicrobial potential of cytarabine. It also indicated that cytarabine inhibited Gram-negative bacteria more profoundly when compared with Gram-positive bacteria in a dose-dependent manner. Cytarabine was also effective against the drug-resistant bacteria both alone as well as in combination with other antibiotics.
Asunto(s)
Acinetobacter baumannii/enzimología , Biopelículas/efectos de los fármacos , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/genética , Acinetobacter baumannii/química , Acinetobacter baumannii/genética , Antibacterianos/química , Antibacterianos/farmacología , Sitios de Unión , Hidrolasas de Éster Carboxílico/farmacología , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Citarabina/química , Escherichia coli/genética , Dominios Proteicos , ARN de Transferencia/química , ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/genética , Especificidad por Sustrato , Propiedades de SuperficieRESUMEN
BACKGROUND & AIMS: Hyperbilirubinemia and hypoalbuminemia are features of hepatic dysfunction that associate with disease severity. This is because hepatic insufficiency causes hypoalbuminemia, which indirectly increases the circulating levels of free bilirubin. Circular dichroism (CD) spectroscopy can be used to quantify the molecular ellipticity (ME) of the albumin-bilirubin complex, and might associate with the severity or outcome of severe alcoholic hepatitis (SAH). METHODS: We performed a cross-sectional study of 265 patients with SAH admitted in the Department of Hepatology, Institute of Liver and Biliary Sciences in New Delhi, India from January 2014 through January 2016. Blood samples were collected and patients were followed for 12 months or death. The molar ratios of bilirubin: albumin and albumin-bilirubin complexes were determined for a discovery cohort (30 patients who survived the study period and 60 patients who did not survive) and compared with those of 60 patients with alcoholic cirrhosis and 30 healthy individuals (controls). Optical activities of albumin-bilirubin complexes in blood samples were determined by CD spectroscopy and compared among groups. Findings were validated in a separate cohort of 150 patients with SAH from the same institute. We studied the correlation between ME and albumin binding capacity (ABiC). RESULTS: The molar ratio of bilirubin: albumin was higher in patients with SAH than with alcoholic cirrhosis or controls (P < .05). Patients with SAH had different CD spectra and higher ME than the other groups (P < .01); ME correlated with model for end-stage liver disease score (with and without Na) and discriminant function (r2 > .3; P < .01). ME values above a cut off of 1.84 mdeg predicted 3-month mortality in patients with SAH with an area under receiver operating characteristic curve of 0.87 (95% CI, 0.79-0.95), a 77% positive predictive value, and a 90% negative predictive value. The hazard ratio and concordance index of ME values for 3-month mortality in patients with SAH was 10% higher than the hazard ratio and concordance index of model for end-stage liver disease score. In patients with SAH, there was an inverse correlation between ME and ABiC (r2 > 0.7; P < .01). We observed a significant reduction in ABiC with increasing levels of bilirubin in vitro prepared albumin-bilirubin complex. CONCLUSION: In a cross-sectional study of patients with SAH, we associated ME of the albumin-bilirubin complex, measured by CD spectroscopy, with outcomes of patients with SAH. Increased loading of bilirubin on albumin could explain reduced albumin function. Bilirubin removal by albumin dialysis might benefit patients with SAH.
Asunto(s)
Bilirrubina/química , Hepatitis Alcohólica/mortalidad , Hepatitis Alcohólica/patología , Albúmina Sérica Humana/química , Adulto , Anciano , Dicroismo Circular , Estudios Transversales , Femenino , Humanos , India , Masculino , Persona de Mediana Edad , Conformación Proteica , Análisis de SupervivenciaRESUMEN
Albumin is a potent scavenger of reactive oxygen species (ROS). However, modifications in albumin structure may reduce its antioxidant properties and modulate its immune-regulatory functions. We examined alterations in circulating albumin in severe alcoholic hepatitis (SAH) patients and their contribution to neutrophil activation, intracellular stress, and alteration in associated molecular pathways. Albumin modifications and plasma oxidative stress were assessed in SAH patients (n = 90), alcoholic cirrhosis patients (n = 60), and healthy controls (n = 30) using liquid chromatography/mass spectrometry and spectrophotometry. Activation and intracellular ROS were measured in healthy neutrophils after treatment with purified albumin from the study groups. Gene expression of SAH neutrophils was analyzed and compared to gene expression from healthy neutrophils after stimulation with purified albumin from SAH patient plasma. SAH-albumin showed the highest albumin oxidative state (P < 0.05) and prominent alteration as human nonmercaptalbumin 2 (P < 0.05). Plasma oxidative stress (advanced oxidative protein product) was higher in SAH versus alcoholic cirrhosis patients and healthy controls (P < 0.05). Neutrophil gelatinase-associated lipocalin, myeloperoxidase, and intracellular ROS levels were highest in SAH-albumin-treated neutrophils (P < 0.05). Genes associated with neutrophil activation, ROS production, intracellular antioxidation, and leukocyte migration plus genes for proinflammatory cytokines and various toll-like receptors were overexpressed in SAH neutrophils compared to healthy neutrophils (P < 0.05). Expression of the above-mentioned genes in SAH-albumin-stimulated healthy neutrophils was comparable with SAH patient neutrophils, except for genes associated with apoptosis, endoplasmic reticulum stress, and autophagy (P < 0.05). CONCLUSIONS: In patients with SAH, there is a significant increase in albumin oxidation, and albumin acts as a pro-oxidant; this promotes oxidative stress and inflammation in SAH patients through activation of neutrophils. (Hepatology 2017;65:631-646).
Asunto(s)
Hepatitis Alcohólica/sangre , Hepatitis Alcohólica/patología , Estrés Oxidativo/fisiología , Especies Reactivas de Oxígeno/sangre , Adulto , Cromatografía Liquida , Estudios Transversales , Femenino , Humanos , Lipocalina 2/metabolismo , Pruebas de Función Hepática , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Activación Neutrófila , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Valores de Referencia , Índice de Severidad de la Enfermedad , Estadísticas no ParamétricasRESUMEN
Secretory signalling glycoprotein (SPX-40) from mammary gland is highly expressed during involution. This protein is involved in a programmed cell death during tissue remodelling which occurs at the end of lactation. SPX-40 was isolated and purified from buffalo (SPB-40) from the samples obtained during involution. One solution of SPB-40 was made by dissolving it in buffer containing 25â¯mM Tris-HCl and 50â¯mM NaCl at pH 8.0. Another solution was made by adding 25% ethanol to the above solution. The biological effects of SPB-40 dissolved in above two solutions were evaluated on MCF-7 breast cancer cell lines. Free SPB-40 indicated significant pro-apoptotic effects while ethanol exposed SPB-40 showed considerably reduced effects on the apoptosis. SPB-40 was crystallized in the native state. The crystals of SPB-40 were soaked in four separate solutions containing 25% acetone, 25% ethanol, 25% butanol and 25% MPD. Four separate data sets were collected and their structures were determined at high resolutions. In all the four structures, the molecules of acetone, ethanol, butanol and MPD respectively were observed in the hydrophobic binding pocket of SPB-40. As a result of which, the conformation of Trp78 was altered thus blocking the binding site in SPB-40 leading to the loss of activity.
Asunto(s)
Apoptosis/efectos de los fármacos , Glicoproteínas/farmacología , Glándulas Mamarias Animales/química , Transducción de Señal/efectos de los fármacos , Animales , Búfalos , Cristalografía por Rayos X , Femenino , Glicoproteínas/química , Glicoproteínas/aislamiento & purificación , Glicoproteínas/metabolismo , Humanos , Lactancia/metabolismo , Células MCF-7 , Glándulas Mamarias Animales/metabolismo , Relación Estructura-ActividadRESUMEN
Maltodextrin glucosidase (MalZ) hydrolyses short malto-oligosaccharides from the reducing end releasing glucose and maltose in Escherichia coli. MalZ is a highly aggregation prone protein and molecular chaperonins GroEL and GroES assist in the folding of this protein to a substantial level. The N-terminal region of this enzyme appears to be a unique domain as seen in sequence comparison studies with other amylases as well as through homology modelling. The sequence and homology model analysis show a probability of disorder in the N-Terminal region of MalZ. The crystal structure of this enzyme has been reported in the present communication. Based on the crystallographic structure, it has been interpreted that the N-terminal region of the enzyme (Met1-Phe131) might be unstructured or flexible. To understand the role of the N-terminal region of MalZ in its enzymatic activity, and overall stability, a truncated version (Ala111-His616) of MalZ was created. The truncated version failed to fold into an active enzyme both in E. coli cytosol and in vitro even with the assistance of chaperonins GroEL and GroES. Furthermore, the refolding effort of N-truncated MalZ in the presence of isolated N-terminal domain didn't succeed. Our studies suggest that while the structural rigidity or orientation of the N-terminal region of the MalZ protein may not be essential for its stability and function, but the said domain is likely to play an important role in the formation of the native structure of the protein when present as an integral part of the protein.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/química , Glucósidos/química , Glicósido Hidrolasas/química , Secuencia de Aminoácidos , Sitios de Unión , Chaperonina 60/química , Chaperonina 60/genética , Chaperonina 60/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Glucósidos/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Modelos Moleculares , Agregado de Proteínas , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología Estructural de Proteína , Especificidad por SustratoRESUMEN
Lactoperoxidase (LPO) belongs to mammalian heme peroxidase superfamily, which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO), and thyroid peroxidase (TPO). LPO catalyzes the oxidation of a number of substrates including thiocyanate while TPO catalyzes the biosynthesis of thyroid hormones. LPO is also been shown to catalyze the biosynthesis of thyroid hormones indicating similar functional and structural properties. The binding studies showed that 2-mercaptoimidazole (MZY) bound to LPO with a dissociation constant of 0.63 µM. The inhibition studies showed that the value of IC50 was 17 µM. The crystal structure of the complex of LPO with MZY showed that MZY bound to LPO in the substrate-binding site on the distal heme side. MZY was oriented in the substrate-binding site in such a way that the sulfur atom is at a distance of 2.58 Å from the heme iron. Previously, a similar compound, 3-amino-1,2,4-triazole (amitrole) was also shown to bind to LPO in the substrate-binding site on the distal heme side. The amino nitrogen atom of amitrole occupied the same position as that of sulfur atom in the present structure indicating a similar mode of binding. Recently, the structure of the complex of LPO with a potent antithyroid drug, 1-methylimidazole-2-thiol (methimazole, MMZ) was also determined. It showed that MMZ bound to LPO in the substrate-binding site on the distal heme side with 2 orientations. The position of methyl group was same in the 2 orientations while the positions of sulfur atom differed indicating a higher preference for a methyl group.
Asunto(s)
Etilenotiourea/análogos & derivados , Lactoperoxidasa/química , Hormonas Tiroideas/química , Sitios de Unión , Cristalografía por Rayos X , Etilenotiourea/química , Etilenotiourea/metabolismo , Hemo/química , Hemo/metabolismo , Humanos , Lactoperoxidasa/metabolismo , Metimazol/química , Metimazol/uso terapéutico , Conformación Proteica , Especificidad por Sustrato , Azufre , Glándula Tiroides/química , Glándula Tiroides/enzimología , Hormonas Tiroideas/biosíntesisRESUMEN
Proliferating cell nuclear antigen (PCNA) acts as a sliding clamp to support DNA replication and repair. The structure of PCNA from Leishmania donovani (LdPCNA) has been determined at 2.73Å resolution. Structure consists of six crystallographically independent molecules which form two trimeric rings. The pore diameter of the individual trimeric ring is of the order of 37Å. The two rings are stacked through their front to front faces. In order to gain a stable packing, the rings are rotated by 42° about the pore axis and shifted by 7Å and tilted by 16° along the perpendicular direction to pore axis. This form of stacking reduced the effective diameter of the pore to 32Å. The sequence of LdPCNA consists of a long segment of 41 amino acid residues (186-Gly-Val-Ser-Asp-Arg-Ser-Thr-Lys-Ser-Glu-Val-Lys-Ala-Glu-Val-Lys-Ala-Glu-Ala-Arg-Asp-Asp-Asp-Glu-Glu-Pro-Leu-Ser-Arg-Lys-Tyr-Gly-Lys-Ala-Asp-Ser-Ser-Ala-Asn-Ala-Ile-226) whereas the corresponding segments in other PCNAs contain only eight residues corresponding to 186-Gly-Val-Ser-Asp-Arg------224-Asn-Ala-Ile-226. The enhanced length of this segment in LdPCNA may influence its mode of interaction with DNA and other proteins. The dissociation constants obtained using real time binding studies with surface plasmon resonance (SPR) for two peptides, Lys-Arg-Arg-Gln-Thr-Ser-Met-Thr-Asp-Phe-Tyr-His (P1) from human cyclin-dependent kinase inhibitor-1(CKI-1) and Lys-Thr-Gln-Gly-Arg-Leu-Asp-Ser-Phe-Phe-Thr-Val (P2) from flap endonuclease 1 (Fen-1) as well as with two small molecule inhibitors, (S)-4-(4-(2-amino-3-hydroxypropyl)-2, 6-diiodophenoxy) phenol hydrochloride (ADPH) and N-(3-methylthiophene-2-carboxylicacid)-N'-((3-hydroxy-2-naphthalenyl) methylene) hydrazide (MCMH) are 0.29±0.09µM, 0.37±0.08µM, 0.35±0.09µM and 1.20±0.08µM respectively. The corresponding values obtained using fluorescence spectroscopic methods were 0.22±0.06µM, 0.68±0.07µM, 0.44±0.07µM and 0.75±0.05µM respectively.
Asunto(s)
ADN Protozoario/química , Leishmania donovani/química , Antígeno Nuclear de Célula en Proliferación/química , Proteínas Protozoarias/química , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/química , ADN Protozoario/genética , ADN Protozoario/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Endonucleasas de ADN Solapado/química , Expresión Génica , Leishmania donovani/metabolismo , Modelos Moleculares , Fenoles/química , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Lactoperoxidase (LPO) is a member of mammalian heme peroxidase superfamily whose other members are myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). In these enzymes, the heme moiety is linked to protein through two or three covalent bonds. In the mature LPO, the heme moiety is linked to protein through two ester bonds with highly conserved glutamate and aspartate residues. The previously reported structures of LPO have confirmed the formation of two covalent linkages involving Glu258 and Asp108 with 1-methyl and 5-methyl groups of pyrrole rings A and C respectively. We report here a new form of structure of LPO where the covalent bond between Glu258 and 1-methyl group of pyrrole ring A is present only in a fraction of protein molecules. In this case, the side chain of Glu258 occupies two distinct positions, each of which has a 0.5 occupancy. In one position, it forms a normal ester covalent linkage while in the second position, the side chain of Glu258 is located in the middle of the substrate binding site on the distal heme side. In this position, the atom of the side chain of Glu258 forms several contacts with atoms of other residues and heme moiety. Out of the two observed positions of the side chain of Glu258, the former contributes to the stabilization of heme position and improved catalytic action of LPO while the latter is responsible for the reduced stability of the heme position as well as it blocks the substrate binding site.
Asunto(s)
Hemo/metabolismo , Lactoperoxidasa/metabolismo , Animales , Ácido Aspártico/metabolismo , Sitios de Unión/fisiología , Bovinos , Cristalografía por Rayos X/métodos , Ácido Glutámico/metabolismo , Mamíferos/metabolismo , Modelos Moleculares , Peroxidasa/metabolismo , Conformación ProteicaRESUMEN
Phospholipase A2 (PLA2) catalyzes the hydrolysis of phospholipids into arachidonic acid and lysophospholipids. Arachidonic acid is used as a substrate in the next step of the multistep pathway leading to the production of eicosanoids. The eicosanoids, in extremely low concentrations, are required in a number of physiological processes. However, the increase in their concentrations above the essential physiological requirements leads to various inflammatory conditions. In order to prevent the unwanted rise in the concentrations of eicosanoids, the actions of PLA2 and other enzymes of the pathway need to be blocked. We report here the structures of five complexes of group IIA PLA2 from Daboia russelli pulchella with tightly binding inhibitors, (i) p-coumaric acid, (ii) resveratrol, (iii) spermidine, (iv) corticosterone and (v) gramine derivative. The binding studies using fluorescence spectroscopy and surface plasmon resonance techniques for the interactions of PLA2 with the above five compounds showed high binding affinities with values of dissociation constants (KD) ranging from 3.7×10(-8) M to 2.1×10(-9) M. The structure determinations of the complexes of PLA2 with the above five compounds showed that all the compounds bound to PLA2 in the substrate binding cleft. The protein residues that contributed to the interactions with these compounds included Leu2, Leu3, Phe5, Gly6, Ile9, Ala18, Ile19, Trp22, Ser23, Cys29, Gly30, Cys45, His48, Asp49 and Phe106. The positions of side chains of several residues including Leu2, Leu3, Ile19, Trp31, Lys69, Ser70 and Arg72 got significantly shifted while the positions of active site residues, His48, Asp49, Tyr52 and Asp99 were unperturbed.
Asunto(s)
Inhibidores de Fosfolipasa A2/metabolismo , Fosfolipasas A2/química , Fosfolipasas A2/metabolismo , Animales , Sitios de Unión , Cristalografía por Rayos X , Cinética , Ligandos , Sustancias Macromoleculares/química , Modelos Moleculares , Inhibidores de Fosfolipasa A2/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Daboia , ViperidaeRESUMEN
The bilobal lactoferrin is an approximately 76 kDa glycoprotein. It sequesters two Fe(3+) ions together with two CO(3)(2-) ions. The C-terminal half (residues, Tyr342-Arg689, C-lobe) of bovine lactoferrin (BLF) (residues Ala1-Arg689) was prepared by limited proteolysis using trypsin. Both C-lobe and intact BLF were saturated to 100%. Both of them retained up to nearly 85% of iron at pH 6.5. At pH 5.0, C-lobe retained 75% of iron whereas intact protein could retain only slightly more than 60%. At pH 4.0 both contained 25% iron and at pH 2.0 they were left with iron concentration of only 10%. The structure of iron saturated C-lobe was determined at 2.79 Å resolution and refined to R(cryst) and R(free) factors of 0.205 and 0.273, respectively. The structure contains two crystallographically independent molecules, A and B. They were found to have identical structures with an r.m.s. shift of 0.5 Å for their C(α) atoms. A high solvent content of 66% was observed in the crystals. The average value of an overall B-factor was 68.0 Å(2). The distance of 2.9 Å observed for the coordination bond between Fe(3+) ion and N(e2) of His595 appeared to be considerably longer than the normally observed values of 1.9-2.2 Å. This indicated that the coordination bond involving His595 may be absent. Other coordination distances were observed in the range of 2.1-2.3 Å. Based on the present structure of iron saturated C-lobe, it may be stated that His595 is the first residue to dissociate from ferric ion when the pH is lowered.
Asunto(s)
Hierro/química , Hierro/metabolismo , Lactoferrina/química , Lactoferrina/metabolismo , Animales , Bovinos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Tripsina/metabolismoRESUMEN
Peptidyl-tRNA hydrolase is an essential enzyme which acts as one of the rescue factors of the stalled ribosomes. It is an esterase that hydrolyzes the ester bond in the peptidyl-tRNA molecules, which are products of ribosome stalling. This enzyme is required for rapid clearing of the peptidyl-tRNAs, the accumulation of which in the cell leads to cell death. Over the recent years, it has been heralded as an attractive drug target for antimicrobial therapeutics. Two distinct classes of peptidyl-tRNA hydrolase, Pth and Pth2, have been identified in nature. This review gives an overview of the structural and functional aspects of Pth, along with its sequence and structural comparison among various species of bacteria. While the mode of binding of the substrate to Pth and the mechanism of hydrolysis are still speculated upon, the structure-based drug design using this protein as the target is still largely unexplored. This review focuses on the structural features of Pth, giving a direction to structure-based drug design on this protein.
Asunto(s)
Bacterias/enzimología , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/metabolismo , Hidrólisis , Especificidad por SustratoRESUMEN
During the course of protein synthesis in the cell, the translation process is often terminated due to various reasons. As a result, peptidyl-tRNA molecules are released which are toxic to the cell as well reducing the availability of free amino acid and tRNA molecules for the required protein synthesis in the cell. Such a situation is corrected by an enzyme, Pth (peptidyl-tRNA hydrolase), which catalyses the release of free tRNA and peptide moieties from peptidyl-tRNAs. This means that the active Pth is essential for the survival of bacteria. In order to design inhibitors of PaPth (Pth from Pseudomonas aeruginosa), we determined the structures of PaPth in its native and bound states with compounds amino acylate-tRNA analogue and 5-azacytidine. The structure determination of the native protein revealed that the substrate-binding site was partially occupied by Glu161 from the neigh-bouring molecule. The structure of PaPth indicated that the substrate-binding site can be broadly divided into three distinct subsites. The structures of the two complexes showed that the amino acylate-tRNA analogue filled three subsites, whereas 5-azacytidine filled two subsites. The common sugar and the base moieties of the two compounds occupied identical positions in the cleft. Using surface plasmon resonance, the dissociation constants for the amino acylate-tRNA analogue and 5-azacytidine were found to be 3.53×10-8 M and 5.82×10-8 M respectively.