RESUMEN
The crystal structure of thermitase, a subtilisin-type serine proteinase from Thermoactinomyces vulgaris, was determined by X-ray diffraction at 1.4 A resolution. The structure was solved by a combination of molecular and isomorphous replacement. The starting model was that of subtilisin BPN' from the Protein Data Bank, determined at 2.5 A resolution. The high-resolution refinement was based on data collected using synchrotron radiation with a Fuji image plate as detector. The model of thermitase refined to a conventional R factor of 14.9% and contains 1997 protein atoms, 182 water molecules and two Ca ions. The tertiary structure of thermitase is similar to that of the other subtilisins although there are some significant differences in detail. Comparison with subtilisin BPN' revealed two major structural differences. The N-terminal region in thermitase, which is absent in subtilisin BPN', forms a number of contacts with the tight Ca2+ binding site and indeed provides the very tight binding of the Ca ion. In thermitase the loop of residues 60 to 65 forms an additional (10) beta-strand of the central beta-sheet and the second Ca2+ binding site that has no equivalent in the subtilisin BPN' structure. The observed differences in the Ca2+ binding and the increased number of ionic and aromatic interactions in thermitase are likely sources of the enhanced stability of thermitase.
Asunto(s)
Endopeptidasas , Serina Endopeptidasas , Secuencia de Aminoácidos , Sitios de Unión , Calcio/metabolismo , Endopeptidasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Ácido Nucleico , Subtilisinas , Difracción de Rayos XRESUMEN
The structures of Penicillium vitale and beef liver catalase have been determined to atomic resolution. Both catalases are tetrameric proteins with deeply buried heme groups. The amino acid sequence of beef liver catalase is known and contains (at least) 506 amino acid residues. Although the sequence of P. vitale catalase has not yet been determined chemically, 670 residues have been built into the 2 A resolution electron density map and have been given tentative assignments. A large portion of each catalase molecule (91% of residues in beef liver catalase and 68% of residues in P. vitale catalase) shows structural homology. The root-mean-square deviation between 458 equivalenced C alpha atoms is 1.17 A. The dissimilar parts include a small fragment of the N-terminal arm and an additional "flavodoxin-like" domain at the carboxy end of the polypeptide chain of P. vitale catalase. In contrast, beef liver catalase contains one bound NADP molecule per subunit in a position equivalent to the chain region, leading to the flavodoxin-like domain, of P. vitale catalase. The position and orientation of the buried heme group in the two catalases, relative to the mutually perpendicular molecular dyad axes, are identical within experimental error. A mostly hydrophobic channel leads to the buried heme group. The surface opening to the channel differs due to the different disposition of the amino-terminal arm and the presence of the additional flavodoxin-like domain in P. vitale catalase. Possible functional implications of these comparisons are discussed.
Asunto(s)
Catalasa , Hígado/enzimología , Penicillium/enzimología , Secuencia de Aminoácidos , Animales , Evolución Biológica , Bovinos , Clostridium/análisis , Cristalografía , Flavodoxina , Hemo , Sustancias Macromoleculares , Modelos Moleculares , NADP , Conformación ProteicaRESUMEN
The three-dimensional structure analysis of crystalline fungal catalase from Penicillium vitale has been extended to 2.0 A resolution. The crystals belong to space group P3(1)21, with the unit cell parameters of a = b = 144.4 A and c = 133.8 A. The asymmetric unit contains half a tetrameric molecule of 222 symmetry. Each subunit is a single polypeptide chain of approximately 670 amino acid residues and binds one heme group. The amino acid sequence has been tentatively determined by computer graphics model building (using the FRODO system) and comparison with the known sequence of beef liver catalase. The atomic model has been refined by the Hendrickson & Konnert (1981) restrained least-squares program against 68,000 reflections between 5 A and 2 A resolution. The final R-factor is 0.31 after 24 refinement cycles. The secondary and tertiary structure of the catalase has been analyzed.
Asunto(s)
Catalasa , Penicillium/enzimología , Secuencia de Aminoácidos , Aminoácidos/análisis , Cristalografía , Hemo , Enlace de Hidrógeno , Sustancias Macromoleculares , Modelos Moleculares , Conformación ProteicaRESUMEN
The leghaemoglobins have oxygen affinities 11 to 24 times higher than that of sperm whale myoglobin, due mainly to higher rates of association. To find out why, we have determined the structures of deoxy- and oxy-leghaemoglobin II of the lupin at 1.7 A resolution. Results confirm the general features found in previous X-ray analyses of this protein. The unique feature that has now emerged is the rotational freedom of the proximal histidine. In deoxy-leghaemoglobin the imidazole oscillates between two alternative orientations, eclipsing either the lines N1-N3 or N2-N4 of the porphyrin; in oxy-leghaemoglobin it is fixed in a staggered orientation. The iron atom moves from a position 0.30 A from the plane of the pyrrole nitrogen atoms in deoxy- to a position in the plane in oxy-leghaemoglobin while the Fe-
Asunto(s)
Fabaceae/química , Leghemoglobina/análogos & derivados , Leghemoglobina/química , Leghemoglobina/metabolismo , Plantas Medicinales , Cristalografía por Rayos X , Globinas/química , Modelos Moleculares , Oxidación-Reducción , Oxígeno/química , Oxígeno/metabolismo , Conformación ProteicaRESUMEN
The structure of the Carnation Mottle Virus (CMtV) capsid protein has been determined at 3.2 A resolution by the method of molecular replacement. Three-dimensional data were collected from a small number of crystals (sp.g. I23, a = 382.6 A) using the synchrotron radiation with an image plate as detector. The coordinates of Tomato Bushy Stunt Virus (TBSV) were used as a searching model. Refinement of the coordinates of 7,479 non-hydrogen atoms performed by the program XPLOR, has led to an R-factor of 18.3%. It was found that the amino acid chain fold of capsid protein is very similar to that in other icosahedral viruses. However, there are some differences in the contact regions between protein subunits and also the lack of the beta-annulus around the 3-fold icosahedral axes. The structural and biochemical results lead us to consider an alternative assembly pathway.
Asunto(s)
Proteínas de la Cápside , Cápside/química , Virus de Plantas/química , Virus ARN/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Datos de Secuencia Molecular , Virus de Plantas/ultraestructura , Conformación Proteica , Virus ARN/ultraestructuraRESUMEN
Crystals of the neurotoxin-I (NTX-I) from the venom of the middle Asian cobra Naja naja oxiana have been grown by vapour diffusion and dialysis methods. The crystals belong to space group P2(1)2(1)2 with dimension of a = 25.19 A, b = 75.59 A, c = 36.09 A and diffract to 1.9 A resolution. The asymmetric unit contains one molecule (Vm = 2.2 A/Da). Using the molecule of alpha-cobratoxin (CTX) as a starting model for NTX-I structure determination coordinates of C alpha atoms of the NTX-I molecule were obtained and the position of NTX-I in the unit cell was derived.
Asunto(s)
Proteínas Neurotóxicas de Elápidos , Venenos Elapídicos , Secuencia de Aminoácidos , Animales , Cristalografía , Datos de Secuencia Molecular , Conformación Proteica , Difracción de Rayos XRESUMEN
The three-dimensional crystal structure of catalase from Micrococcus lysodeikticus has been solved by multiple isomorphous replacement and refined at 1.5 A resolution. The subunit of the tetrameric molecule of 222 symmetry consists of a single polypeptide chain of about 500 amino acid residues and one haem group. The crystals belong to space group P4(2)2(1)2 with unit cell parameters a = b = 106.7 A, c = 106.3 A, and there is one subunit of the tetramer per asymmetric unit. The amino acid sequence has been tentatively determined by computer graphics model building and comparison with the known three-dimensional structure of beef liver catalase and sequences of several other catalases. The atomic model has been refined by Hendrickson and Konnert's least-squares minimisation against 94,315 reflections between 8 A and 1.5 A. The final model consists of 3,977 non-hydrogen atoms of the protein and haem group, 426 water molecules and one sulphate ion. The secondary and tertiary structures of the bacterial catalase have been analyzed and a comparison with the structure of beef liver catalase has been made.
Asunto(s)
Catalasa/química , Micrococcus/enzimología , Secuencia de Aminoácidos , Evolución Biológica , Catalasa/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Difracción de Rayos XRESUMEN
Uridine phosphorylase from E. coli (Upase) has been crystallized using vapor diffusion technique in a new monoclinic crystal form. The structure was determined by the molecular replacement method at 2.5 A resolution. The coordinates of the trigonal crystal form were used as a starting model and the refinement by the program XPLOR led to the R-factor of 18.6%. The amino acid fold of the protein was found to be the same as that in the trigonal crystals. The positions of flexible regions were refined. The conclusion about the involvement in the active site is in good agreement with the results of the biochemical experiments.
Asunto(s)
Escherichia coli/enzimología , Uridina Fosforilasa/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
On the basis of electron microscope data the structure of tubular crystals of catalase has been determined with resolution of approximately 25 A. The symmetry of the helical packing of molecules is 142/17. The three-dimensional reconstruction has been carried out in real space. The catalase molecule consists of four subunits whose centers from a fairly flattened tetrahedron. The molecule has dimensions of 69X87X92 A.
Asunto(s)
Catalasa , Animales , Bovinos , Cristalización , Microscopía Electrónica , Modelos Moleculares , Conformación ProteicaRESUMEN
The crystal structure of the membrane-active antibiotic-cyclopeptide gramicidin S complex with urea was determined by the X-ray structure analysis. The gramicidin S molecule possesses an antiparallel beta-structure, its slightly twisted 30-membered cycle has a roughly rectangular form about 4.8 x 13.6 A in size, with the lesser side being formed by the main chain atoms of Phe and Pro residues. The maximum size of the molecule is 22.9 A. A characteristic feature of the molecule is the position of the extended side chains of the Orn residues on one side of the molecular cycle in the form of peculiar "legs--tentacles". One of these legs is "fastened" by the intramolecular H-bond to O atom of the nearer Phe4 residue, the other being free. The distance between the terminal NE atoms of the Orn residues is 5.7 A. The side chains of the Phe and Orn2 residues have trans-orientation, those of the Val, Orn7, Leu residues gauche-orientation. For Val1 and Leu3 side chains statistical disorder of the terminal C atoms is realized. The pyrrolidine rings of the Pro residues adopt Cs-C beta-exo conformation. There are one urea and 20 water molecules per one antibiotic molecule in the structure. The positions of three water molecules are fully occupied, the others with the probability of 0.56-0.20. One of the "water" positions is occupied on 2/3 by water, and on 1/3 by the O atom of the alcohol. There is a complicated system of intra- and intermolecular H-bonds in the structure, with and without the participation of water, alcohol and urea molecules. The gramicidin S molecules, collecting around 3(1) axis according to the left-handed double helix, form the channels whose outside hydrophobic surface is built of the side uncharged radicals, the inside surface being built of the main chain atoms, mainly of the O and N atoms and of the ornithine "tails" with uncharged NE atoms at the termini. The outer diameter of the channel is 29-43 A, inner (without ornithine "tails") is about 12.7 A. At the expense of the change of these "tails" conformation, the inner diameter of the channel filled with water molecules may change from 3.4 up to 6.3 A. Thus, the ions and particles of a rather large size may pass through the channel. The gramicidin channels are discovered and described for the first time. The channels in the crystal structure are close-packed under the hexagonal law.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Gramicidina/metabolismo , Canales Iónicos/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Membrana Celular/metabolismo , Gramicidina/química , Datos de Secuencia Molecular , Conformación Proteica , Difracción de Rayos XAsunto(s)
Proteínas/análisis , Actinas , Aminoácidos , Aspartato Aminotransferasas , Colágeno , Cristalización , ADN , Leghemoglobina , Microscopía Electrónica , Modelos Biológicos , Modelos Químicos , Conformación Molecular , Conformación de Ácido Nucleico , Pepsina A , Péptidos , ARN , Difracción de Rayos XAsunto(s)
Sustancias Macromoleculares , Microscopía Electrónica , Actinas , Adenoviridae , Animales , Bacteriófagos , Catalasa , Colifagos , Computadores , Eritrocitos/enzimología , Glutamato Deshidrogenasa , Hemocianinas , Hemoglobinas , Herpesviridae , Holografía , Humanos , Hígado/enzimología , Métodos , Microscopía Electrónica de Rastreo , Modelos Estructurales , Miosinas , Virus de Plantas , Poliomavirus , Complejo Piruvato Deshidrogenasa , RibosomasRESUMEN
Aspartate transaminase (EC 2.6.1.1., Asp-transaminase) has been studied extensively, and much is now known about its physico-chemical, catalytic and other properties. X-ray studies that can provide a structural foundation for the events that occur during the transamination reaction are under way on three species of Asp-transaminase: the cytosolic enzyme from pig and chicken hearts, and the mitochondrial chicken heart enzyme. We describe here the interpretation of an electron density map of Asp-transaminase from chicken heart cytosol at 3.5 A.
Asunto(s)
Aspartato Aminotransferasas , Miocardio/enzimología , Animales , Sitios de Unión , Pollos , Citosol/enzimología , Enlace de Hidrógeno , Conformación Proteica , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
Using the solvent-protons' longitudinal magnetic relaxation rates (p.m.r.) for Lupinus luteus leghaemoglobin derivatives the accessibility of the haem has been evaluated by our "stereo-chemical p.m.r. titration" method with nonexchangeable protons of aliphatic lower alcohols in otherwise deuterated solutions. The haem in leghaemoglobin is more accessible and its protein environment more flexible compared with vertebrate haemoglobins. The correlation time in aquometleghaemglobin aqueous solution has been determined by measuring the frequency dispersion of the p.m.r. rates between 6.1 and 93 MHZ. Taking into account the measured value of tauc = (7.7 +/- 0.5 x 10(-10) s the iron-to-proton inter-spin distances have been calculated. The significance of these distances as well as the electronic g-factor anisotrophy for elucidation of fine structural details of the haem-environment are discussed.
Asunto(s)
Hemoproteínas/análisis , Leghemoglobina/análisis , Plantas/análisis , Hemo/análisis , Espectroscopía de Resonancia Magnética , Metahemoglobina/análisis , Protones , TemperaturaRESUMEN
The three-dimensional structure of the manganese-phosphate complex of inorganic pyrophosphatase from Saccharomyces cerevisiae has been refined to an R factor of 19.0% at 2.4-A resolution. X-ray data were collected from a single crystal using an imaging plate scanner and synchrotron radiation. There is one dimeric molecule in the asymmetric unit. The upper estimate of the root-mean-square coordinate error is 0.4 A using either the delta A plot or the superposition of the two crystallographically independent subunits. The good agreement between the coordinates of the two subunits, which were not subjected to non-crystallographic symmetry restraints, provides independent validation of the structure analysis. The active site in each subunit contains four manganese ions and two phosphates. The manganese ions are coordinated by the side chains of aspartate and glutamate residues. The phosphate groups, which were identified on the basis of their local stereochemistry, interact either directly or via water molecules with manganese ions and lysine, arginine, and tyrosine side chains. The phosphates are bridged by two of the manganese ions. The outer phosphate is exposed to solvent. The inner phosphate is surrounded by all four manganese ions. The ion-binding sites are related to the order of binding previously established from kinetic studies. A hypothesis for the transition state of the catalytic reaction is put forward.
Asunto(s)
Manganeso/metabolismo , Fosfatos/metabolismo , Pirofosfatasas/química , Saccharomyces cerevisiae/enzimología , Cristalografía por Rayos X , Hidrólisis , Pirofosfatasa Inorgánica , Conformación Proteica , Pliegue de Proteína , Pirofosfatasas/aislamiento & purificación , Pirofosfatasas/metabolismo , Especificidad por SustratoRESUMEN
Two isoforms of an extracellular endonuclease, nucleases Sm1 and Sm2, were purified from culture fluid of Serratia marcescens strain BIO MI by ligand-exchange chromatography on phosphocellulose and DEAE-Toyopearl 650S. The pI-values for nucleases Sm1 and Sm2 were found to be 7.1 and 6.7, respectively. The amino acid analysis and N-terminal amino acid sequencing of the proteins showed a significant degree of homology between the enzymes. The nuclease Sm1 has been crystallized from ammonium sulfate solution by the vapour diffusion technique. The crystals belong to the space group P2(1)2(1)2(1) with unit cell constants a = 69.0, b = 106.7, c = 74.8 A, contain two molecules in an asymmetric unit, packing density Vm = 2.3 A/Da, and diffract to at least 1.5 A resolution. The Pt- and UO2-derivatives of the protein were obtained. Preliminary X-ray investigation of nuclease Sm2 crystals was carried out.