RESUMEN

The essential helicase loader protein G39P encoded by Bacillus subtilis SPP1 phage has been overproduced in Escherichia coli and purified. The wild-type protein has been crystallized by the hanging-drop vapour-diffusion method in a primitive hexagonal space group, probably P6(1)22/P6(5)22, but the crystals diffract to only 3.4 A and are poorly reproducible. Mass-spectrometric analysis has revealed marked proteolytic cleavage from the C-terminus and the presence of a major species corresponding to deletion of the 14 C-terminal residues. Thus, a new variant of the protein (G39P112) has been engineered that corresponds to a 14-residue C-terminal truncation. The G39P112 variant has also been crystallized but now in a primitive orthorhombic form, probably P2(1)2(1)2 or P2(1)2(1)2(1), with unit-cell parameters a = 85.6, b = 89.7, c = 47.6 A, with diffraction to 2.4 A on a synchrotron source and with greatly improved reproducibility. Calculation of V(M) values for this G39P112 variant suggests the presence of three monomers in the asymmetric unit, corresponding to a solvent content of about 47%. A selenomethionine-incorporated form of the protein has been produced and a full three-wavelength MAD data collection undertaken.

Asunto(s)

Bacillus subtilis/química , Bacteriófagos/enzimología , Proteínas Portadoras/química , Proteínas no Estructurales Virales/química , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/aislamiento & purificación , Clonación Molecular , Cristalización , Péptidos y Proteínas de Señalización Intracelular , Espectrometría de Masas , Proteínas no Estructurales Virales/biosíntesis , Proteínas no Estructurales Virales/aislamiento & purificación

RESUMEN

Asymmetrically cleaving diadenosine 5',5"'-P(1),P(4)-tetraphosphate (Ap4A) hydrolase activity has been detected in extracts of adult Caenorhabditis elegans and the corresponding cDNA amplified and expressed in Escherichia coli. As expected, sequence analysis shows the enzyme to be a member of the Nudix hydrolase family. The purified recombinant enzyme behaves as a typical animal Ap4A hydrolase. It hydrolyses Ap4A with a K(m) of 7 microM and k(cat) of 27 s(-1) producing AMP and ATP as products. It is also active towards other adenosine and diadenosine polyphosphates with four or more phosphate groups, but not diadenosine triphosphate, always generating ATP as one of the products. It is inhibited non-competitively by fluoride (K(i)=25 microM) and competitively by adenosine 5'-tetraphosphate with Ap4A as substrate (K(i)=10 nM). Crystals of diffraction quality with the morphology of rectangular plates were readily obtained and preliminary data collected. These crystals diffract to a minimum d-spacing of 2 A and belong to either space group C222 or C222(1). Phylogenetic analysis of known and putative Ap4A hydrolases of the Nudix family suggests that they fall into two groups comprising plant and Proteobacterial enzymes on the one hand and animal and archaeal enzymes on the other. Complete structural determination of the C. elegans Ap4A hydrolase will help determine the basis of this grouping.

Asunto(s)

Ácido Anhídrido Hidrolasas/genética , Caenorhabditis elegans/genética , Ácido Anhídrido Hidrolasas/biosíntesis , Ácido Anhídrido Hidrolasas/química , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/enzimología , Catálisis , Cromatografía en Gel , Clonación Molecular , Cristalización , ADN Complementario/biosíntesis , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Difracción de Rayos X

RESUMEN

Enoyl acyl carrier protein (ACP) reductase catalyses the last reductive step of fatty acid biosynthesis, reducing the enoyl group of a growing fatty acid chain attached to ACP to its acyl product using NAD(P)H as the cofactor. This enzyme is the target for the diazaborine class of antibacterial agents, the biocide triclosan, and one of the targets for the front-line anti-tuberculosis drug isoniazid. The structures of complexes of Escherichia coli enoyl-ACP reductase (ENR) from crystals grown in the presence of NAD+ and a family of diazaborine compounds have been determined. Analysis of the structures has revealed that a mobile loop in the structure of the binary complex with NAD+ becomes ordered on binding diazaborine/NAD+ but displays a different conformation in the two subunits of the asymmetric unit. The work presented here reveals how, for one of the ordered conformations adopted by the mobile loop, the mode of diazaborine binding correlates well with the activity profiles of the diazaborine family. Additionally, diazaborine binding provides insights into the pocket on the enzyme surface occupied by the growing fatty acid chain.

Asunto(s)

Compuestos de Boro/química , Compuestos de Boro/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/enzimología , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/química , Sitios de Unión , Compuestos de Boro/metabolismo , Cristalografía por Rayos X , Enoil-ACP Reductasa (NADH) , Inhibidores Enzimáticos/farmacología , Modelos Moleculares , NAD/metabolismo , Oxidorreductasas/metabolismo , Unión Proteica , Conformación Proteica , Subunidades de Proteína , Relación Estructura-Actividad , Triclosán/química , Triclosán/metabolismo , Triclosán/farmacología

RESUMEN

BACKGROUND: Glycerol-3-phosphate (1)-acyltransferase(G3PAT) catalyzes the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acyl-CoAs into the sn-1 position of glycerol 3-phosphate to yield 1-acylglycerol-3-phosphate. G3PATs can either be selective, preferentially using the unsaturated fatty acid, oleate (C18:1), as the acyl donor, or nonselective, using either oleate or the saturated fatty acid, palmitate (C16:0), at comparable rates. The differential substrate specificity for saturated versus unsaturated fatty acids seen within this enzyme family has been implicated in the sensitivity of plants to chilling temperatures. RESULTS: The three-dimensional structure of recombinant G3PAT from squash chloroplast has been determined to 1.9 A resolution by X-ray crystallography using the technique of multiple isomorphous replacement and provides the first representative structure of an enzyme of this class. CONCLUSIONS: The tertiary structure of G3PAT comprises two domains, the larger of which, domain II, features an extensive cleft lined by hydrophobic residues and contains at one end a cluster of positively charged residues flanked by a H(X)(4)D motif, which is conserved amongst many glycerolipid acyltransferases. We predict that these hydrophobic and positively charged residues represent the binding sites for the fatty acyl substrate and the phosphate moiety of the glycerol 3-phosphate, respectively, and that the H(X)(4)D motif is a critical component of the enzyme's catalytic machinery.

Asunto(s)

Glicerol-3-Fosfato O-Aciltransferasa/química , Secuencia de Aminoácidos , Sitios de Unión , Glicerofosfatos/química , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Especificidad por Sustrato , Verduras/enzimología

RESUMEN

The LrpA transcriptional regulator from Pyrococcus furiosus, a member of the leucine-responsive regulatory protein (Lrp) family, has been crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. The crystals belong to the tetragonal system and are in space group I4(1)22, with unit-cell parameters a = b = 104.5, c = 245.1 A. Consideration of the values of V(M) and possible packing of the molecules within the cell suggest that the asymmetric unit contains a dimer. Examination of the behaviour of the protein on gel-filtration columns and analysis of the self-rotation function suggests that the molecule is an octamer in solution at around pH 5. Determination of the structure of this protein will provide insights into the mechanisms responsible for DNA-protein recognition at high temperature and into how the regulatory properties of the Lrp family are modified by the presence or absence of effector molecules.

Asunto(s)

Proteínas Arqueales/química , Proteínas de Unión al ADN/química , Pyrococcus furiosus/química , Factores de Transcripción/química , Cristalización , Cristalografía por Rayos X , Proteína Reguladora de Respuesta a la Leucina , Conformación Proteica , Estructura Cuaternaria de Proteína

RESUMEN

Fab I, enoyl acyl carrier protein reductase (ENR), is an enzyme used in fatty acid synthesis. It is a single chain polypeptide in plants, bacteria, and mycobacteria, but is part of a complex polypeptide in animals and fungi. Certain other enzymes in fatty acid synthesis in apicomplexan parasites appear to have multiple forms, homologous to either a plastid, plant-like single chain enzyme or more like the animal complex polypeptide chain. We identified a plant-like Fab I in Plasmodium falciparum and modelled the structure on the Brassica napus and Escherichia coli structures, alone and complexed to triclosan (5-chloro-2-[2,4 dichlorophenoxy] phenol]), which confirmed all the requisite features of an ENR and its interactions with triclosan. Like the remarkable effect of triclosan on a wide variety of bacteria, this compound markedly inhibits growth and survival of the apicomplexan parasites P. falciparum and Toxoplasma gondii at low (i.e. IC50 congruent with150-2000 and 62 ng/ml, respectively) concentrations. Discovery and characterisation of an apicomplexan Fab I and discovery of triclosan as lead compound provide means to rationally design novel inhibitory compounds.

Asunto(s)

Antimaláricos/farmacología , Oxidorreductasas/antagonistas & inhibidores , Plasmodium falciparum/efectos de los fármacos , Toxoplasma/efectos de los fármacos , Triclosán/farmacología , Secuencia de Aminoácidos , Animales , Enoil-ACP Reductasa (NADH) , Inhibidores Enzimáticos/farmacología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Oxidorreductasas/química , Plasmodium falciparum/enzimología , Plasmodium falciparum/crecimiento & desarrollo , Alineación de Secuencia , Toxoplasma/enzimología , Toxoplasma/crecimiento & desarrollo

RESUMEN

The LrpA protein from the hyperthermophilic archaeon Pyrococcus furiosus belongs to the Lrp/AsnC family of transcriptional regulatory proteins, of which the Escherichia coli leucine-responsive regulatory protein is the archetype. Its crystal structure has been determined at 2.9 A resolution and is the first for a member of the Lrp/AsnC family, as well as one of the first for a transcriptional regulator from a hyperthermophile. The structure consists of an N-terminal domain containing a helix-turn-helix (HtH) DNA-binding motif, and a C-terminal domain of mixed alpha/beta character reminiscent of a number of RNA- and DNA-binding domains. Pyrococcus furiosus LrpA forms a homodimer mainly through interactions between the antiparallel beta-sheets of the C-terminal domain, and further interactions lead to octamer formation. The LrpA structure suggests how the protein might bind and possibly distort its DNA substrate through use of its HtH motifs and control gene expression. A possible location for an effector binding site is proposed by using sequence comparisons with other members of the family coupled to mutational analysis.

Asunto(s)

Proteínas Arqueales/química , Proteínas de Unión al ADN/química , Pyrococcus furiosus/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Sitios de Unión , Rastreo Diferencial de Calorimetría , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Dimerización , Proteínas de Escherichia coli , Regulación de la Expresión Génica Arqueal , Leucina/genética , Leucina/metabolismo , Proteína Reguladora de Respuesta a la Leucina , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Pyrococcus furiosus/genética , Alineación de Secuencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

BACKGROUND: beta-Keto acyl carrier protein reductase (BKR) catalyzes the pyridine-nucleotide-dependent reduction of a 3-oxoacyl form of acyl carrier protein (ACP), the first reductive step in de novo fatty acid biosynthesis and a reaction often performed in polyketide biosynthesis. The Brassica napus BKR enzyme is NADPH-dependent and forms part of a dissociable type II fatty acid synthetase (FAS). Significant sequence similarity is observed with enoyl acyl carrier protein reductase (ENR), the other reductase of FAS, and the short-chain alcohol dehydrogenase (SDR) family. RESULTS: The first crystal structure of BKR has been determined at 2.3 A resolution in a binary complex with an NADP(+) cofactor. The structure reveals a homotetramer in which each subunit has a classical dinucleotide-binding fold. A triad of Ser154, Tyr167 and Lys171 residues is found at the active site, characteristic of the SDR family. Overall BKR has a very similar structure to ENR with good superimposition of catalytically important groups. Modelling of the substrate into the active site of BKR indicates the need for conformational changes in the enzyme. CONCLUSIONS: A catalytic mechanism can be proposed involving the conserved triad. Helix alpha6 must shift its position to permit substrate binding to BKR and might act as a flexible lid on the active site. The similarities in fold, mechanism and substrate binding between BKR, which catalyzes a carbon-oxygen double-bond reduction, and ENR, the carbon-carbon double-bond oxidoreductase in FAS, suggest a close evolutionary link during the development of the fatty acid biosynthetic pathway.

Asunto(s)

Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Brassica/enzimología , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa , Adenina/química , Adenina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Evolución Molecular , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Cuaternaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad por Sustrato

RESUMEN

The NADP-dependent beta-keto acyl-carrier protein reductase (BKR) from Brassica napus has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol of average molecular weight 1500 as the precipitant. The crystals belong to the hexagonal space group P6(4)22, with unit-cell parameters a = b = 129. 9, c = 93.1 A, alpha = beta = 90, gamma = 120 degrees. Calculated values for V(m), the use of rotation and translation functions and consideration of the packing suggest that the asymmetric unit contains a monomer. The crystals diffract to beyond 2.8 A resolution and are more amenable to X-ray diffraction analysis than those reported previously for the Escherichia coli enzyme. The structure determination of B. napus BKR will provide important insights into the catalytic mechanism of the enzyme and into the evolution of the fatty-acid elongation cycle by comparisons with the other oxidoreductase of the pathway, enoyl acyl-carrier protein reductase (ENR).

Asunto(s)

Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/aislamiento & purificación , Brassica/enzimología , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa , Oxidorreductasas de Alcohol/genética , Brassica/genética , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación

RESUMEN

Molecular genetic studies with strains of Escherichia coli resistant to triclosan, an ingredient of many anti-bacterial household goods, have suggested that this compound works by acting as an inhibitor of enoyl reductase (ENR) and thereby blocking lipid biosynthesis. We present structural analyses correlated with inhibition data, on the complexes of E. coli and Brassica napus ENR with triclosan and NAD(+) which reveal how triclosan acts as a site-directed, picomolar inhibitor of the enzyme by mimicking its natural substrate. Elements of both the protein and the nucleotide cofactor play important roles in triclosan recognition, providing an explanation for the factors controlling its tight binding to the enzyme and for the emergence of triclosan resistance.

Asunto(s)

Antiinfecciosos Locales/química , Antiinfecciosos Locales/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Triclosán/química , Triclosán/metabolismo , Sitios de Unión , Compuestos de Boro/metabolismo , Brassica/química , Cristalografía por Rayos X , Enoil-ACP Reductasa (NADH) , Inhibidores Enzimáticos/metabolismo , Escherichia coli/enzimología , Modelos Moleculares , Oxidorreductasas/antagonistas & inhibidores , Proteínas de Plantas/química , Conformación Proteica

RESUMEN

Enoyl acyl carrier protein reductase (ENR) is involved in fatty acid biosynthesis. In Escherichia coli this enzyme is the target for the experimental family of antibacterial agents, the diazaborines, and for triclosan, a broad spectrum antimicrobial agent. Biochemical studies have suggested that the mechanism of diazaborine inhibition is dependent on NAD(+) and not NADH, and resistance of Brassica napus ENR to diazaborines is thought to be due to the replacement of a glycine in the active site of the E. coli enzyme by an alanine at position 138 in the plant homologue. We present here an x-ray analysis of crystals of B. napus ENR A138G grown in the presence of either NAD(+) or NADH and the structures of the corresponding ternary complexes with thienodiazaborine obtained either by soaking the drug into the crystals or by co-crystallization of the mutant with NAD(+) and diazaborine. Analysis of the ENR A138G complex with diazaborine and NAD(+) shows that the site of diazaborine binding is remarkably close to that reported for E. coli ENR. However, the structure of the ternary ENR A138G-NAD(+)-diazaborine complex obtained using co-crystallization reveals a previously unobserved conformational change affecting 11 residues that flank the active site and move closer to the nicotinamide moiety making extensive van der Waals contacts with diazaborine. Considerations of the mode of substrate binding suggest that this conformational change may reflect a structure of ENR that is important in catalysis.

Asunto(s)

Brassica/enzimología , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Enoil-ACP Reductasa (NADH) , Escherichia coli/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , NAD/metabolismo , Conformación Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido

Asunto(s)

Antiinfecciosos Locales/farmacología , Inhibidores Enzimáticos/farmacología , Oxidorreductasas/antagonistas & inhibidores , Triclosán/farmacología , Antiinfecciosos Locales/química , Catálisis , Farmacorresistencia Microbiana , Enoil-ACP Reductasa (NADH) , Inhibidores Enzimáticos/química , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Modelos Moleculares , Imitación Molecular , Oxidorreductasas/genética , Triclosán/química

RESUMEN

During homologous recombination in Escherichia coli the RuvA, B and C proteins interact specifically with the Holliday junction formed by the action of RecA to promote the strand-exchange reaction. RuvA, a homotetrameric protein of molecular weight 88 kDa, has been overexpressed in E. coli, purified and co-crystallized with a synthetic Holliday junction substrate made from four 18-base deoxyoligonucleotides. Crystals were grown using the hanging-drop vapour-diffusion method with sodium acetate as the precipitant. The crystals diffract to a resolution of 6 A and belong to the monoclinic system, space group C2, with cell parameters a = 148, b = 148, c = 106 A and beta = 123 degrees. The X-ray analysis of these crystals should reveal the structure of the Holliday junction and its mode of binding to RuvA, providing new insights into the molecular mechanism of genetic recombination.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , ADN Helicasas , ADN Bacteriano/química , ADN Bacteriano/aislamiento & purificación , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas Bacterianas/genética , Secuencia de Bases , Cristalización , ADN Bacteriano/genética , Proteínas de Unión al ADN/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Conformación Proteica , Recombinación Genética , Difracción de Rayos X

RESUMEN

Diazaborine and isoniazid are, at first sight, unrelated anti-bacterial agents that inhibit the enoyl-ACP reductase (ENR) of Escherichia coli and Mycobacterium tuberculosis respectively. The crystal structures of these enzymes including that of the diazaborine-inhibited E. coli ENR have been obtained at high resolution. Site-directed mutagenesis was used to study the importance of amino acid residues in diazaborine susceptibility and enzyme function. The results show that drug binding and inhibition require the presence of a glycine residue at position 93 of E. coli ENR or at the structurally equivalent position in the plant homologue, which is naturally resistant to the drug. The data confirm the hypothesis that any amino acid side-chain other than hydrogen at this position within the three-dimensional structure of these enzymes will affect diazaborine resistance by encroaching into the drug binding site. Substitutions of Gly-93 by amino acids with small side-chains, such as serine, alanine, cysteine and valine, hardly affected the catalytic parameters and rendered the bacterial host resistant to the drug. Larger amino acid side-chains, such as that of arginine, histidine, lysine and glutamine, completely inactivated the activity of the enzyme.

Asunto(s)

Antibacterianos/farmacología , Compuestos de Boro/farmacología , Inhibidores Enzimáticos/farmacología , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/genética , Alelos , Sustitución de Aminoácidos , Antibacterianos/metabolismo , Catálisis , Farmacorresistencia Microbiana , Enoil-ACP Reductasa (NADH) , Inhibidores Enzimáticos/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Mutagénesis Sitio-Dirigida , Oxidorreductasas/metabolismo , Análisis de Secuencia de ADN

RESUMEN

The NADP-dependent beta-keto acyl carrier protein reductase (BKR) from E. coli has been crystallized by the hanging-drop method of vapour diffusion using poly(ethylene glycol) of average molecular weight 1450. The crystals belong to the hexagonal space group P6122 or P6522 with unit-cell dimensions a = b = 67.8, c = 355.8 A. Calculated values for Vm and consideration of the packing suggest that the asymmetric unit contains a dimer. BKR catalyses the first reductive step in the elongation cycle of fatty-acid biosynthesis. It shares extensive sequence homology with the enzyme which catalyzes the second reductive step in the cycle, enoyl acyl carrier protein reductase (ENR), and thus provides an opportunity to study the evolution of enzyme function in a metabolic pathway. The structure determination will permit the analysis of the molecular basis of its catalytic mechanism and substrate specificity.

Asunto(s)

Oxidorreductasas de Alcohol/química , Escherichia coli/enzimología , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa , Cristalización , Difusión , Programas Informáticos , Difracción de Rayos X

RESUMEN

The diazaborine family of compounds have antibacterial properties against a range of gram-negative bacteria. Initially, this was thought to be due to the prevention of lipopolysaccharide synthesis. More recently, the molecular target of diazaborines has been identified as the NAD(P)H-dependent enoyl acyl carrier protein reductase (ENR), which catalyses the last reductive step of fatty acid synthase. ENR from Mycobacterium tuberculosis is the target for the front-line antituberculosis drug isoniazid. The emergence of isoniazid resistance strains of M. tuberculosis, a chronic infectious disease that already kills more people than any other infection, is currently causing great concern over the prospects for its future treatment, and it has reawakened interest in the mechanism of diazaborine action. Diazaborines only inhibit ENR in the presence of the nucleotide cofactor, and this has been explained through the analysis of the x-ray crystallographic structures of a number of Escherichia coli ENR-NAD+-diazaborine complexes that showed the formation of a covalent bond between the boron atom in the diazaborines and the 2'-hydroxyl of the nicotinamide ribose moiety that generates a noncovalently bound bisubstrate analogue. The similarities in catalytic chemistry and in the conformation of the nucleotide cofactor across the wider family of NAD(P)-dependent oxidoreductases suggest that there are generic opportunities to mimic the interactions seen here in the rational design of bisubstrate analogue inhibitors for other NAD(P)H-dependent oxidoreductases.

Asunto(s)

Antibacterianos/farmacología , Compuestos de Boro/farmacología , Compuestos Heterocíclicos/farmacología , Antibacterianos/química , Compuestos de Boro/química , Resistencia a Medicamentos/genética , Compuestos Heterocíclicos/química , Mutación , Relación Estructura-Actividad

RESUMEN

Here we present the crystal structure of the Escherichia coli protein RuvA bound to a key DNA intermediate in recombination, the Holliday junction. The structure, solved by isomorphous replacement and density modification at 6 A resolution, reveals the molecular architecture at the heart of the branch migration and resolution reactions required to process Holliday intermediates into recombinant DNA molecules. It also reveals directly for the first time the structure of the Holliday junction. A single RuvA tetramer is bound to one face of a junction whose four DNA duplex arms are arranged in an open and essentially four-fold symmetric conformation. Protein-DNA contacts are mediated by two copies of a helix-hairpin-helix motif per RuvA subunit that contact the phosphate backbone in a very similar manner. The open structure of the junction stabilized by RuvA binding exposes a DNA surface that could be bound by the RuvC endonuclease to promote resolution.

Asunto(s)

Proteínas Bacterianas/química , ADN Helicasas , Proteínas de Unión al ADN/química , ADN/química , Conformación de Ácido Nucleico , Proteínas Bacterianas/metabolismo , Composición de Base , Secuencia de Bases , Cristalización , Cristalografía por Rayos X , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli , Proteínas de Escherichia coli , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica

RESUMEN

Comparison of the structure of Escherichia coli RuvA with other proteins in the Protein Data Bank gives insights into the probable modes of association of RuvA with the Holliday junction during homologous recombination. All three domains of the RuvA protein possess striking structural similarities to other DNA-binding proteins. Additionally, the second domain of RuvA contains two copies of the helix-hairpin-helix (HhH) structural motif, which has been implicated in non-sequence-specific DNA binding. The two copies of the motif are related by approximate 2-fold symmetry and may form a bidentate DNA-binding module. The results described provide support for the organization of the arms of the DNA in our RuvA/Holliday junction complex model and support the involvement of the HhH motifs in DNA binding.

Asunto(s)

Proteínas Bacterianas/química , ADN Helicasas , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/química , Escherichia coli/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , ADN Polimerasa beta/química , ADN Polimerasa beta/metabolismo , ADN Bacteriano/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Conformación de Ácido Nucleico , Conformación Proteica , Homología de Secuencia de Aminoácido , Polimerasa Taq

RESUMEN

Enoyl acyl carrier protein reductase catalyses the last reductive step of fatty acid biosynthesis, reducing an enoyl acyl carrier protein to an acyl-acyl carrier protein with NAD(P)H as the cofactor. The crystal structure of enoyl reductase (ENR) from Escherichia coli has been determined to 2.1 A resolution using a combination of molecular replacement and isomorphous replacement and refined using data from 10 A to 2.1 A to an R-factor of 0.16. The final model consists of the four subunits of the tetramer, wherein each subunit is composed of 247 of the expected 262 residues, and a NAD+ cofactor for each subunit of the tetramer contained in the asymmetric unit plus a total of 327 solvent molecules. There are ten disordered residues per subunit which form a loop near the nucleotide binding site which may become ordered upon substrate binding. Each monomer is composed of a seven-stranded parallel beta-sheet flanked on each side by three alpha-helices with a further helix lying at the C terminus of the beta-sheet. This fold is highly reminiscent of the Rossmann fold, found in many NAD(P)H-dependent enzymes. Analysis of the sequence and structure of ENR and comparisons with the family of short-chain alcohol dehydrogenases, identify a conserved tyrosine and lysine residue as important for catalytic activity. Modelling studies suggest that a region of the protein surface that contains a number of strongly conserved hydrophobic residues and lies adjacent to the nicotinamide ring, forms the binding site for the fatty acid substrate.

Asunto(s)

Escherichia coli/enzimología , Ácido Graso Sintasas/química , Ácido Graso Sintasas/metabolismo , NAD/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Alcohol Deshidrogenasa/química , Alcohol Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Brassica/enzimología , Secuencia Conservada , Cristalografía por Rayos X , Enoil-ACP Reductasa (NADH) , Ácidos Grasos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mycobacterium tuberculosis/enzimología , NAD/química , Nucleótidos/metabolismo , Conformación Proteica , Pliegue de Proteína , Homología de Secuencia de Aminoácido , Especificidad por Sustrato

RESUMEN

The E. coli protein RuvA (resistance to ultraviolet light) has been overexpressed in E. coli, purified and crystallized using the hanging-drop vapour-diffusion method with sodium chloride as the precipitant. The crystals, which diffract to beyond 1.9 A, belong to the tetragonal system, space group P4 with unit-cell dimensions of a = 83.7, c = 33.1 A with a monomer in the asymmetric unit. RuvA is known to be a tetramer and thus the crystal symmetry implies that its quaternary structure will be based on fourfold rotation symmetry rather than 222 symmetry. This is consistent with electron microscopy data on Holliday junction DNA complexes and implies that the arms of the four DNA duplexes involved in recombination adopt fourfold rotation symmetry.