RESUMEN

Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization.

Asunto(s)

Isoantígenos/química , Proteínas de Plasma Seminal/química , Animales , Cristalización , Escherichia coli , Femenino , Isoantígenos/metabolismo , Ratones , Conformación Molecular , Proteínas Recombinantes/metabolismo , Proteínas de Plasma Seminal/metabolismo

RESUMEN

The isochorismate synthase DhbC from Bacillus anthracis is essential for the biosynthesis of the siderophore bacillibactin by this pathogenic bacterium. The structure of the selenomethionine-substituted protein was determined to 2.4 Šresolution using single-wavelength anomalous diffraction. B. anthracis DhbC bears the strongest resemblance to the Escherichia coli isochorismate synthase EntC, which is involved in the biosynthesis of another siderophore, namely enterobactin. Both proteins adopt the characteristic fold of other chorismate-utilizing enzymes, which are involved in the biosynthesis of various products, including siderophores, menaquinone and tryptophan. The conservation of the active-site residues, as well as their spatial arrangement, suggests that these enzymes share a common Mg(2+)-dependent catalytic mechanism.

Asunto(s)

Bacillus anthracis/química , Proteínas Bacterianas/química , Transferasas Intramoleculares/química , Magnesio/química , Oligopéptidos/química , Sideróforos/química , Secuencia de Aminoácidos , Bacillus anthracis/enzimología , Bacillus anthracis/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cationes Bivalentes , Secuencia Conservada , Cristalografía por Rayos X , Enterobactina/biosíntesis , Enterobactina/química , Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/genética , Transferasas Intramoleculares/metabolismo , Magnesio/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Oligopéptidos/biosíntesis , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Selenometionina/química , Selenometionina/metabolismo , Sideróforos/biosíntesis , Homología Estructural de Proteína

RESUMEN

Anabolic ornithine transcarbamoylase (aOTC) catalyzes the reaction between carbamoyl phosphate (CP) and L-ornithine (ORN) to form L-citrulline and phosphate in the urea cycle and L-arginine biosynthesis. The crystal structure of unliganded aOTC from Campylobacter jejuni (Cje aOTC) was determined at 2.7 Å resolution and refined to an R(work) of 20.3% and an R(free) of 24.0%. Cje aOTC is a trimer that forms a head-to-head pseudohexamer in the asymmetric unit. Each monomer is composed of an N-terminal CP-binding domain and a C-terminal ORN-binding domain joined by two interdomain helices. The Cje aOTC structure presents an open conformation of the enzyme with a relatively flexible orientation of the ORN-binding domain respective to the CP-binding domain. The conformation of the B2-H3 loop (residues 68-78), which is involved in binding CP in an adjacent subunit of the trimer, differs from that seen in homologous proteins with CP bound. The loop containing the ORN-binding motif (DxxxSMG, residues 223-230) has a conformation that is different from those observed in unliganded OTC structures from other species, but is similar to those in structures with bound ORN analogs. The major differences in tertiary structure between Cje aOTC and human aOTC are described.

Asunto(s)

Campylobacter jejuni/enzimología , Ornitina Carbamoiltransferasa/química , Secuencia de Aminoácidos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología Estructural de Proteína

RESUMEN

New protocols and instrumentation significantly boost the outcome of structural biology, which has resulted in significant growth in the number of deposited Protein Data Bank structures. However, even an enormous increase of the productivity of a single step of the structure determination process may not significantly shorten the time between clone and deposition or publication. For example, in a medium size laboratory equipped with the LabDB and HKL-3000 systems, we show that automation of some (and integration of all) steps of the X-ray structure determination pathway is critical for laboratory productivity. Moreover, we show that the lag period after which the impact of a technology change is observed is longer than expected.

Asunto(s)

Automatización de Laboratorios , Cristalografía por Rayos X , Proteínas/química , Bases de Datos de Proteínas , Conformación Proteica , Difracción de Rayos X

RESUMEN

We determined the 1.6-A resolution crystal structure of a conserved hypothetical 29.9-kDa protein from the SIGY-CYDD intergenic region encoded by a Bacillus subtilis open reading frame in the YXKO locus. YXKO homologues are broadly distributed and are by and large described as proteins with unknown function. The YXKO protein has an alpha/beta fold and shows high structural homology to the members of a ribokinase-like superfamily. However, YXKO is the only member of this superfamily known to form tetramers. Putative binding sites for adenosine triphosphate (ATP), a substrate, and Mg(2+)-binding sites were revealed in the structure of the protein, based on high structural similarity to ATP-dependent members of the superfamily. Two adjacent monomers contribute residues to the active site. The crystal structure provides valuable information about the YXKO protein's tertiary and quaternary structure, the biochemical function of YXKO and its homologues, and the evolution of its ribokinase-like superfamily.

Asunto(s)

Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Evolución Molecular , Humanos , Metales/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Conformación Proteica , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad

RESUMEN

Lipoxygenases are an important class of non-heme iron enzymes that catalyze the hydroperoxidation of unsaturated fatty acids. The details of the enzymatic mechanism of lipoxygenases are still not well understood. This study utilizes a combination of kinetic and structural probes to relate the lipoxygenase mechanism of action with structural modifications of the iron's second coordination sphere. The second coordination sphere consists of Gln(495) and Gln(697), which form a hydrogen bond network between the substrate cavity and the first coordination sphere (Asn(694)). In this investigation, we compared the kinetic and structural properties of four mutants (Q495E, Q495A, Q697N, and Q697E) with those of wild-type soybean lipoxygenase-1 and determined that changes in the second coordination sphere affected the enzymatic activity by hydrogen bond rearrangement and substrate positioning through interaction with Gln(495). The nature of the C-H bond cleavage event remained unchanged, which demonstrates that the mutations have not affected the mechanism of hydrogen atom tunneling. The unusual and dramatic inverse solvent isotope effect (SIE) observed for the Q697E mutant indicated that an Fe(III)-OH(-) is the active site base. A new transition state model for hydrogen atom abstraction is proposed.

Asunto(s)

Glycine max/enzimología , Lipooxigenasa/química , Lipooxigenasa/genética , Mutagénesis Sitio-Dirigida , Proteínas de Hierro no Heme/química , Proteínas de Hierro no Heme/genética , Sustitución de Aminoácidos/genética , Unión Competitiva/genética , Dicroismo Circular , Cristalografía por Rayos X , Óxido de Deuterio/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Glutamina/química , Glutamina/genética , Cinética , Lipooxigenasa/metabolismo , Proteínas de Hierro no Heme/metabolismo , Oxidación-Reducción , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Solventes , Glycine max/genética , Espectrofotometría Ultravioleta , Relación Estructura-Actividad , Especificidad por Sustrato/genética , Viscosidad

RESUMEN

Rubidium is a monovalent metal that can be used as a counterion in protein solutions. X-ray anomalous scattering from rubidium ions bound to the protein surface was used for phasing of the crystal structure of the hsp60 apical domain from Thermus thermophilus. Multiple-wavelength anomalous dispersion (MAD) data were collected from a crystal obtained from a solution containing 0.2 M rubidium salt. One molecule of protein (147 amino acids) binds one well ordered and one poorly ordered Rb atom. Phases calculated with the program SHARP were sufficient for automatic tracing and side-chain assignment using the program ARP/wARP. The data show that bound rubidium ions can be used to determine protein structures and to study the interaction of monovalent metal ions with proteins and other macromolecules.

Asunto(s)

Proteínas Bacterianas/química , Rubidio/química , Chaperonina 60/química , Modelos Moleculares , Propiedades de Superficie , Thermus thermophilus/química

RESUMEN

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms while raltitrexed (Tomudex, ZD1694) is an antifolate inhibitor of TS approved for clinical use in several European countries. The crystal structure of the complex between recombinant human TS, dUMP, and raltitrexed has been determined at 1.9 A resolution. In contrast to the situation observed in the analogous complex of the rat TS, the enzyme is in the closed conformation and a covalent bond between the catalytic Cys 195 and dUMP is present in both subunits. This mode of ligand binding is similar to that of the analogous complex of the Escherichia coli enzyme. The only major differences observed are a direct hydrogen bond between His 196 and the O4 atom of dUMP and repositioning of the side chain of Tyr 94 by about 2 A. The thiophene ring of the drug is disordered between two parallel positions.

Asunto(s)

Nucleótidos de Desoxiuracil/química , Inhibidores Enzimáticos/química , Antagonistas del Ácido Fólico/química , Quinazolinas/química , Tiofenos/química , Timidilato Sintasa/antagonistas & inhibidores , Timidilato Sintasa/química , Sitios de Unión , Simulación por Computador , Cristalización , Cristalografía por Rayos X , Nucleótidos de Desoxiuracil/metabolismo , Dimerización , Humanos , Ligandos , Sustancias Macromoleculares , Modelos Moleculares , Conformación Proteica , Quinazolinas/metabolismo , Tiofenos/metabolismo , Timidilato Sintasa/metabolismo

RESUMEN

Thymidylate synthase (TS) is a major target in the chemotherapy of colorectal cancer and some other neoplasms. The emergence of resistance to the treatment is often related to the increased levels of TS in cancer cells, which have been linked to the elimination of TS binding to its own mRNA upon drug binding, a feedback regulatory mechanism, and/or to the increased stability to intracellular degradation of TS.drug complexes (versus unliganded TS). The active site loop of human TS (hTS) has a unique conformation resulted from a rotation by 180 degrees relative to its orientation in bacterial TSs. In this conformation, the enzyme must be inactive, because the catalytic cysteine is no longer positioned in the ligand-binding pocket. The ordered solvent structure obtained from high resolution crystallographic data (2.0 A) suggests that the inactive loop conformation promotes mRNA binding and intracellular degradation of the enzyme. This hypothesis is supported by fluorescence studies, which indicate that in solution both active and inactive forms of hTS are present. The binding of phosphate ion shifts the equilibrium toward the inactive conformation; subsequent dUMP binding reverses the equilibrium toward the active form. Thus, TS inhibition via stabilization of the inactive conformation should lead to less resistance than is observed with presently used drugs, which are analogs of its substrates, dUMP and CH(2)H(4)folate, and bind in the active site, promoting the active conformation. The presence of an extension at the N terminus of native hTS has no significant effect on kinetic properties or crystal structure.

Asunto(s)

Resistencia a Antineoplásicos/genética , Timidilato Sintasa/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Sitios de Unión , Unión Competitiva , Neoplasias Colorrectales/tratamiento farmacológico , Cristalografía por Rayos X , Cisteína/química , ADN/metabolismo , Nucleótidos de Desoxiuracil/metabolismo , Activación Enzimática , Humanos , Cinética , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Pliegue de Proteína , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Espectrometría de Fluorescencia , Termodinámica

RESUMEN

Formyltetrahydrofolate synthetase (FTHFS) from the thermophilic homoacetogen, Moorella thermoacetica, has an optimum temperature for activity of 55-60 degrees C and requires monovalent cations for both optimal activity and stabilization of tetrameric structure at higher temperatures. The crystal structures of complexes of FTHFS with cesium and potassium ions were examined and monovalent cation binding positions identified. Unexpectedly, NH(4)(+) and K(+), both of which are strongly activating ions, bind at a different site than a moderately activating ion, Cs(+), does. Neither binding site is located in the active site. The sites are 7 A apart, but in each of them, the side chain of Glu 98, which is conserved in all known bacterial FTHFS sequences, participates in metal ion binding. Other ligands in the Cs(+) binding site are four oxygen atoms of main chain carbonyls and water molecules. The K(+) and NH(4)(+) binding site includes the carboxylate of Asp132 in addition to Glu98. Mutant FTHFS's (E98Q, E98D, and E98S) were obtained and analyzed using differential scanning calorimetry to examine the effect of these mutations on the thermostability of the enzyme with and without added K(+) ions. The addition of 0.2 M K(+) ions to the wild-type enzyme resulted in a 10 degrees C increase in the thermal denaturation temperature. No significant increase was observed in E98D or E98S. The lack of a significant effect of monovalent cations on the stability of E98D and E98S indicates that this alteration of the binding site eliminates cation binding. The thermal denaturation temperature of E98Q was 3 degrees C higher than that of the wild-type enzyme in the absence of the cation, indicating that the removal of the unbalanced, buried charge of Glu98 stabilizes the enzyme. These results confirm that Glu98 is a crucial residue in the interaction of monovalent cations with FTHFS.

Asunto(s)

Cationes Monovalentes/química , Formiato-Tetrahidrofolato Ligasa/química , Ácido Aspártico/genética , Sitios de Unión/genética , Rastreo Diferencial de Calorimetría , Cesio/química , Clostridium/enzimología , Cristalografía por Rayos X , Estabilidad de Enzimas/genética , Formiato-Tetrahidrofolato Ligasa/genética , Formiato-Tetrahidrofolato Ligasa/aislamiento & purificación , Ácido Glutámico/genética , Glutamina/genética , Mutagénesis Sitio-Dirigida , Potasio/química , Desnaturalización Proteica , Compuestos de Amonio Cuaternario/química , Termodinámica

RESUMEN

The role of Ser 167 of Escherichia coli thymidylate synthase (TS) in catalysis has been characterized by kinetic and crystallographic studies. Position 167 variants including S167A, S167N, S167D, S167C, S167G, S167L, S167T, and S167V were generated by site-directed mutagenesis. Only S167A, S167G, S167T, and S167C complemented the growth of thymidine auxotrophs of E. coli in medium lacking thymidine. Steady-state kinetic analysis revealed that mutant enzymes exhibited k(cat) values 1.1-95-fold lower than that of the wild-type enzyme. Relative to wild-type TS, K(m) values of the mutant enzymes for 2'-deoxyuridylate (dUMP) were 5-90 times higher, while K(m) values for 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were 1.5-16-fold higher. The rate of dehalogenation of 5-bromo-2'-deoxyuridine 5'-monophosphate (BrdUMP), a reaction catalyzed by TS that does not require CH(2)H(4)folate as cosubstrate, by mutant TSs was analyzed and showed that only S167A and S167G catalyzed the dehalogenation reaction and values of k(cat)/K(m) for the mutant enzymes were decreased by 10- and 3000-fold, respectively. Analysis of pre-steady-state kinetics of ternary complex formation revealed that the productive binding of CH(2)H(4)folate is weaker to mutant TSs than to the wild-type enzyme. Chemical transformation constants (k(chem)) for the mutant enzymes were lower by 1.1-6.0-fold relative to the wild-type enzyme. S167A, S167T, and S167C crystallized in the I2(1)3 space group and scattered X-rays to either 1.7 A (S167A and S167T) or 2.6 A (S167C). The high-resolution data sets were refined to a R(crys) of 19.9%. In the crystals some cysteine residues were derivatized with 2-mercaptoethanol to form S,S-(2-hydroxyethyl)thiocysteine. The pattern of derivatization indicates that in the absence of bound substrate the catalytic cysteine is not more reactive than other cysteines. It is proposed that the catalytic cysteine is activated by substrate binding by a proton-transfer mechanism in which the phosphate group of the nucleotide neutralizes the charge of Arg 126', facilitating the transfer of a proton from the catalytic cysteine to a His 207-Asp 205 diad via a system of ordered water molecules.

Asunto(s)

Cisteína/genética , Escherichia coli/enzimología , Timidilato Sintasa/química , Sitios de Unión , Cristalografía por Rayos X , Cisteína/metabolismo , Nucleótidos de Desoxiuracil/metabolismo , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Especificidad por Sustrato , Tetrahidrofolatos/metabolismo , Timidilato Sintasa/genética

Asunto(s)

Cristalografía por Rayos X/métodos , Proteínas/química , Sincrotrones , Cristalografía por Rayos X/instrumentación , Internet , Proyectos de Investigación , Procesamiento de Señales Asistido por Computador

RESUMEN

The structure was solved at 2.5 A resolution using multiwavelength anomalous dispersion (MAD) scattering by Se-Met residues. The subunit of N(10)-formyltetrahydrofolate synthetase is composed of three domains organized around three mixed beta-sheets. There are two cavities between adjacent domains. One of them was identified as the nucleotide binding site by homology modeling. The large domain contains a seven-stranded beta-sheet surrounded by helices on both sides. The second domain contains a five-stranded beta-sheet with two alpha-helices packed on one side while the other two are a wall of the active site cavity. The third domain contains a four-stranded beta-sheet forming a half-barrel. The concave side is covered by two helices while the convex side is another wall of the large cavity. Arg 97 is likely involved in formyl phosphate binding. The tetrameric molecule is relatively flat with the shape of the letter X, and the active sites are located at the end of the subunits far from the subunit interface.

Asunto(s)

Clostridium/enzimología , Formiato-Tetrahidrofolato Ligasa/química , Secuencia de Aminoácidos , Clostridium/química , Cristalización , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia

RESUMEN

Drug-resistant variants of thymidylate synthase (TS) can potentially be used in gene therapy applications to decrease the myelosuppressive side effects of TS-directed anticancer agents or to select genetically modified cells in vivo. Mutations of proline 303 of human TS confer resistance to TS-directed fluoropyrimidines and antifolates (). We generated the corresponding variants in Escherichia coli TS (ecTS), position 254, to better understand the mechanism by which mutations at this residue confer resistance. In addition, because ecTS is intrinsically resistant to several antifolates when compared with human TS, we suspected that greater resistance could be achieved with the bacterial enzyme. The P254L enzyme conferred >100-fold resistance to both raltitrexed and 5-fluoro-2'-deoxyuridine (FdUrd) compared with wild-type ecTS. Four additional mutants (P254F, P254S, P254G, and P254D), each of which complemented growth of a TS-deficient cell line, were generated, isolated, and characterized. Steady-state values of K(m) for dUMP and k(cat) were not substantially different among the variants and were comparable with the wild-type values, but K(m) for methylenetetrahydrofolate (CH(2)H(4)PteGlu) was >10-fold higher for P254D. Values of k(on) and k(off) for nucleotide binding, which were obtained by stopped-flow spectroscopy, were virtually unchanged among the mutants. Drastic differences were observed for CH(2)H(4)PteGlu binding, with K(d) values >15-fold higher than observed with the wild-type enzyme; surprisingly, the proposed isomerization reaction that is very evident for the wild-type enzyme is not observed with P254S. The decrease in affinity for CH(2)H(4)PteGlu correlates well with K(i) values obtained for three TS-directed inhibitors. These results show that mutations at Pro-254 specifically affect the initial binding interactions between enzyme and cofactor and also alter the ability of the mutant enzymes to undergo conformational changes that occur on ternary complex formation. The crystal structure of P254S was determined at 1.5 A resolution and is the most precise structure of TS available. When compared with wild-type TS, the structure shows local conformational changes affecting mostly Asp-253; its carbonyl is rotated approximately 40 degrees, and the side chain forms an ion pair with Arg-225.

Asunto(s)

Escherichia coli/enzimología , Antagonistas del Ácido Fólico/farmacología , Timidilato Sintasa/metabolismo , Sustitución de Aminoácidos , Cristalografía por Rayos X , Nucleótidos de Desoxiuracil/farmacología , Resistencia a Medicamentos , Farmacorresistencia Microbiana/fisiología , Inhibidores Enzimáticos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Fluorodesoxiuridilato/farmacología , Humanos , Cinética , Mutación , Prolina/metabolismo , Conformación Proteica , Quinazolinas/farmacología , Tetrahidrofolatos/farmacología , Timidilato Sintasa/antagonistas & inhibidores , Timidilato Sintasa/química , Timidilato Sintasa/genética , Transfección

RESUMEN

Lipoxygenases catalyze the biosynthesis of leukotrienes, lipoxins, and other lipid-derived mediators that are involved in a wide variety of pathophysiological processes, including inflammation, allergy, and tumorigenesis. Mammalian lipoxygenases are activated by a calcium-mediated translocation to intracellular membranes upon cell stimulation, and cooperate with cytosolic phospholipase A2 at the membrane surface to generate eicosanoids. Although it has been documented that plant cell stimulation increases intracellular Ca2+ concentration and activates cytosolic phospholipase A2, followed by lipoxygenase-catalyzed conversion of the liberated linolenic acid to jasmonic acid, no evidence is available for Ca2+-regulated membrane binding and activity of plant lipoxygenases. Plant lipoxygenases, unlike their mammalian counterparts, are believed to function independently of calcium or membranes. Here we present spectroscopic evidence for a calcium-regulated membrane-binding mechanism of soybean lipoxygenase-1 (L-1). Both calcium and membrane binding affect the structure and the mode of action of L-1. Free L-1 in solution is less accessible to the polar solvent and converts linoleic acid to conjugated dienes, whereas surface binding increases solvent accessibility and stimulates conjugated ketodiene production. Calcium exerts a biphasic effect on the structure and activity of L-1. Our results uncover a new regulatory mechanism for plant lipoxygenases and delineate common features in animal and plant cell signaling pathways.

Asunto(s)

Proteínas de Unión al Calcio/metabolismo , Glycine max/enzimología , Lipooxigenasa/metabolismo , Adsorción , Secuencia de Aminoácidos , Calcio/metabolismo , Calcio/farmacología , Proteínas de Unión al Calcio/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Transferencia de Energía , Activación Enzimática/efectos de los fármacos , Lipooxigenasa/química , Modelos Moleculares , Datos de Secuencia Molecular , Fosfatidilcolinas/farmacología , Fosfolípidos/farmacología , Unión Proteica/efectos de los fármacos , Espectrometría de Fluorescencia , Espectroscopía Infrarroja por Transformada de Fourier

RESUMEN

BACKGROUND: Neutral lipases are ubiquitous and diverse enzymes. The molecular architecture of the structurally characterized lipases is similar, often despite a lack of detectable homology at the sequence level. Some of the microbial lipases are evolutionarily related to physiologically important mammalian enzymes. For example, limited sequence similarities were recently noted for the Streptomyces exfoliatus lipase (SeL) and two mammalian platelet-activating factor acetylhydrolases (PAF-AHs). The determination of the crystal structure of SeL allowed us to explore the structure-function relationships in this novel family of homologous hydrolases. RESULTS: The crystal structure of SeL was determined by multiple isomorphous replacement and refined using data to 1.9 A resolution. The molecule exhibits the canonical tertiary fold of an alpha/beta hydrolase. The putative nucleophilic residue, Ser131, is located within a nucleophilic elbow and is hydrogen bonded to His209, which in turn interacts with Asp177. These three residues create a triad that closely resembles the catalytic triads found in the active sites of other neutral lipases. The mainchain amides of Met132 and Phe63 are perfectly positioned to create an oxyanion hole. Unexpectedly, there are no secondary structure elements that could render the active site inaccessible to solvent, like the lids that are commonly found in neutral lipases. CONCLUSIONS: The crystal structure of SeL reinforces the notion that it is a homologue of the mammalian PAF-AHs. We have used the catalytic triad in SeL to model the active site of the PAF-AHs. Our model is consistent with the site-directed mutagenesis studies of plasma PAF-AH, which implicate Ser273, His351 and Asp296 in the active site. Our study therefore provides direct support for the hypothesis that the plasma and isoform II PAF-AHs are triad-containing alpha/beta hydrolases.

Asunto(s)

Fosfolipasas A/química , Streptomyces/enzimología , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Secuencia de Aminoácidos , Sitios de Unión/fisiología , Cristalografía por Rayos X , Proteínas Fúngicas/química , Enlace de Hidrógeno , Lipasa/química , Modelos Moleculares , Datos de Secuencia Molecular , Factor de Activación Plaquetaria/fisiología , Estructura Secundaria de Proteína , Alineación de Secuencia , Relación Estructura-Actividad

RESUMEN

Enolase, a glycolytic enzyme that catalyzes the dehydration of 2-phospho-d-glycerate (PGA) to form phosphoenolpyruvate (PEP), is a homodimer in all eukaryotes and many prokaryotes. Here, we report the crystal structure of a complex between yeast enolase and an equilibrium mixture of PGA and PEP. The structure has been refined using 29 854 reflections with an F/sigma(F) of >/=3 to an R of 0.137 with average deviations of bond lengths and bond angles from ideal values of 0.013 A and 3.1 degrees , respectively. In this structure, the dimer constitutes the crystallographic asymmetric unit. The two subunits are similar, and their superposition gives a rms distance between Calpha atoms of 0.91 A. The exceptions to this are the catalytic loop Val153-Phe169 where the atomic positions in the two subunits differ by up to 4 A and the loop Ser250-Gln277, which follows the catalytic loop Val153-Phe169. In the first subunit, the imidazole side chain of His159 is in contact with the phosphate group of the substrate/product molecule; in the other it is separated by water molecules. A series of hydrogen bonds leading to a neighboring enolase dimer can be identified as being responsible for ordering and stabilization of the conformationally different subunits in the crystal lattice. The electron density present in the active site suggests that in the active site with the direct ligand-His159 hydrogen bond PGA is predominantly bound while in the active site where water molecules separate His159 from the ligand the binding of PEP dominates. The structure indicates that the water molecule hydrating carbon-3 of PEP in the PEP --> PGA reaction is activated by the carboxylates of Glu168 and Glu211. The crystals are unique because they have resolved two intermediates on the opposite sides of the transition state.

Asunto(s)

Fosfoenolpiruvato/química , Fosfopiruvato Hidratasa/química , Cristalización , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología

RESUMEN

RhoA, a ubiquitous intracellular GTPase, mediates cytoskeletal responses to extracellular signals. A 2.1 A resolution crystal structure of the human RhoA-GDP complex shows unique stereochemistry in the switch I region, which results in a novel mode of Mg2+ binding.

Asunto(s)

Proteínas de Unión al GTP/química , Guanosina Difosfato/química , Conformación Proteica , Cristalografía por Rayos X , Proteínas de Unión al GTP/metabolismo , Guanosina Difosfato/metabolismo , Humanos , Magnesio/química , Modelos Moleculares , Proteína de Unión al GTP rhoA

Asunto(s)

Difracción de Rayos X/métodos , Difracción de Rayos X/estadística & datos numéricos , Cristalografía por Rayos X/estadística & datos numéricos , Interpretación Estadística de Datos , Programas Informáticos

RESUMEN

Lipoxygenases, which are widely distributed among plant and animal species, are Fe-containing dioxygenases that act on lipids containing (Z,Z)-pentadiene moieties in the synthesis of compounds with a variety of functions. Utilizing an improved strategy of data collection, low temperature, and synchrotron radiation of short wavelength, the structure of ferrous soybean lipoxygenase L-1, a single chain protein of 839 amino acid residues, has been determined by X-ray crystallography to a resolution of 1.4 A. The R-factor for the refined model is 19.7%. General features of the protein structure were found to be consistent with the results of prior crystallographic studies at lower (2.6 A) resolution. In contrast to the prior studies, the binding of a water molecule to the active site Fe was established. The octahedral coordination sphere of the Fe also includes the side chains of His499, His504, His690, and Asn694 as well as the terminal carboxylate of Ile839, which binds as a monodentate ligand. Asn694 is involved in a number of labile polar interactions with other protein groups, including an amide-aromatic hydrogen bond, and appears to be a weak ligand. Several possible access routes for dioxygen and fatty acids to the internal active site and substrate binding cavity are described. The protein structure restricts access to the Fe site such that the formation of an organo-Fe intermediate seems improbable. Structural restrictions pertinent to other proposed reaction intermediates, such as planar pentadienyl and nonplanar allyl radicals, are also discussed.

Asunto(s)

Glycine max/enzimología , Lipooxigenasa/química , Cristalografía por Rayos X , Enlace de Hidrógeno , Hierro/química , Lipooxigenasa/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Especificidad por Sustrato , Agua/química