RESUMEN
The shikimate pathway is essential for survival of the apicomplexan parasites Plasmodium falciparum, Toxoplasma gondii and Cryptosporidium parvum. As it is absent in mammals it is a promising therapeutic target. Herein, we describe the genes encoding the shikimate pathway enzymes in T. gondii. The molecular arrangement and phylogeny of the proteins suggests homology with the eukaryotic fungal enzymes, including a pentafunctional AROM. Current rooting of the eukaryotic evolutionary tree infers that the fungi and apicomplexan lineages diverged deeply, suggesting that the arom is an ancient supergene present in early eukaryotes and subsequently lost or replaced in a number of lineages.
Asunto(s)
Células Eucariotas/enzimología , Evolución Molecular , Genes Protozoarios , Transducción de Señal/genética , Toxoplasma/genética , Oxidorreductasas de Alcohol/genética , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , FilogeniaRESUMEN
We are studying two enzymes from the shikimate pathway, dehydroquinate synthase (DHQS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Both enzymes have been the subject of numerous studies to elucidate their reaction mechanisms. Crystal structures of DHQS and EPSPS in the presence and absence of substrates, cofactors and/or inhibitors are now available. These structures reveal movements of domains, rearrangements of loops and changes in side-chain positions necessary for the formation of a catalytically competent active site. The potential for using complementary small-angle X-ray scattering (SAXS) studies to confirm the presence of these structural differences in solution has also been explored. Comparative analysis of crystal structures, in the presence and absence of ligands, has revealed structural features critical for substrate-binding and catalysis. We have also analysed these structures by generating GRID energy maps to detect favourable binding sites. The combination of X-ray crystallography, SAXS and computational techniques provides an enhanced analysis of structural features important for the function of these complex enzymes.
Asunto(s)
Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/metabolismo , Liasas de Fósforo-Oxígeno/química , Liasas de Fósforo-Oxígeno/metabolismo , Ácido Shikímico/metabolismo , 3-Fosfoshikimato 1-Carboxiviniltransferasa , Sitios de Unión , Modelos Moleculares , Conformación Proteica , Difracción de Rayos XRESUMEN
The background and current context of work on the shikimate-pathway enzymes as potential targets for anti-bacterial, anti-fungal and anti-parasitic drugs is reviewed. Recent work on the third enzyme of the pathway, dehydroquinase, which occurs in two structurally and mechanistically distinct forms, is used to illustrate the present state of studies into rational drug design.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Hidroliasas/metabolismo , Ácido Shikímico/metabolismo , Bacterias/efectos de los fármacos , Bacterias/enzimología , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Hidroliasas/antagonistas & inhibidores , Hidroliasas/química , Plantas/enzimologíaRESUMEN
Shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase. In this paper we have explored the roles of residues in the P-loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins. In common with many members of the P-loop family, shikimate kinase contains a cysteine residue 2 amino acids upstream of the essential lysine residue; the side chains of these residues are shown to form an ion pair. The C13S mutant of shikimate kinase was found to be enzymatically active, whereas the K15M mutant was inactive. However, the latter mutant had both increased thermostability and affinity for ATP when compared to the wild-type enzyme. The structure of the K15M mutant protein has been determined at 1.8 A, and shows that the organization of the P-loop and flanking regions is heavily disturbed. This indicates that, besides its role in catalysis, the P-loop lysine also has an important structural role. The structure of the K15M mutant also reveals that the formation of an additional arginine/aspartate ion pair is the most likely reason for its increased thermostability. From studies of ligand binding it appears that, like adenylate kinase, shikimate kinase binds substrates randomly and in a synergistic fashion, indicating that the two enzymes have similar catalytic mechanisms.
Asunto(s)
Lisina/química , Lisina/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/farmacología , Adenosina Trifosfato/metabolismo , Arginina/química , Ácido Aspártico/química , Sitios de Unión , Rastreo Diferencial de Calorimetría , Catálisis , Dicroismo Circular , Cristalografía por Rayos X , Dickeya chrysanthemi/química , Disulfuros/farmacología , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Oxidantes/farmacología , Unión Proteica , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Reactivos de Sulfhidrilo/farmacología , Temperatura , Ácido Trinitrobencenosulfónico/farmacología , Rayos UltravioletaRESUMEN
QUTR (qutR-encoded transcription-repressing protein) is a multi-domain repressor protein active in the signal-transduction pathway that regulates transcription of the quinic acid utilization (qut) gene cluster in Aspergillus nidulans. In the presence of quinate, production of mRNA from the eight genes of the qut pathway is stimulated by the activator protein QUTA (qutA-encoded transcription-activating protein). Mutations in the qutR gene alter QUTR function such that the transcription of the qut gene cluster is permanently on (constitutive phenotype) or is insensitive to the presence of quinate (super-repressed phenotype). These mutant phenotypes imply that the QUTR protein plays a key role in signal recognition and transduction, and we have used deletion analysis to determine which regions of the QUTR protein are involved in these functions. We show that the QUTR protein recognizes and binds to the QUTA protein in vitro and that the N-terminal 88 amino acids of QUTR are sufficient to inactivate QUTA function in vivo. Deletion analysis and domain-swap experiments imply that the two C-terminal domains of QUTR are mainly involved in signal recognition.
Asunto(s)
Proteínas Represoras/metabolismo , Transducción de Señal , Secuencia de Bases , Sitios de Unión , Cartilla de ADN , Mutagénesis Sitio-Dirigida , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genéticaRESUMEN
Mechanistic differences between type I and type II dehydroquinases have been exploited in the design of type specific inhibitors. (2R)-2-Bromo-3-dehydroquinic acid (3), (2R)-2-fluoro-3-dehydroquinic acid (5) and 2-bromo-3-dehydroshikimic acid (4), all excellent substrates for type II dehydroquinase, are shown to be irreversible inhibitors of type I dehydroquinase.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Hidroliasas/antagonistas & inhibidores , Hidroliasas/metabolismo , Ácido Quínico/análogos & derivados , Ácido Shikímico/análogos & derivados , Escherichia coli/enzimología , Mycobacterium tuberculosis/enzimología , Ácido Quínico/síntesis química , Ácido Quínico/farmacología , Ácido Shikímico/síntesis química , Ácido Shikímico/metabolismo , Ácido Shikímico/farmacologíaRESUMEN
Shikimate dehydrogenase from Escherichia coli has been crystallized by the vapour-diffusion method using ammonium sulfate as a precipitant. Mass spectrometry confirmed the purity of the enzyme and dynamic light scattering was used to find the appropriate additives to yield a monodisperse enzyme solution. The crystals are monoclinic, space group C2, with unit-cell parameters a = 110.0, b = 139.8, c = 102.6 A, beta = 122.2 degrees (at 100 K). Native crystals diffract to 2.3 A in-house on a rotating-anode X-ray source. The asymmetric unit is likely to contain four molecules, related by 222 symmetry, corresponding to a packing density of 2.86 A(3) Da(-1).
Asunto(s)
Oxidorreductasas de Alcohol/química , Escherichia coli/enzimología , Oxidorreductasas de Alcohol/aislamiento & purificación , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Espectrometría de Masas , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
(6R)- and (6S)-6-Fluoro-3-dehydroquinic acids are shown to be substrates for type I and type II dehydroquinases. Their differential reactivity provides insight into details of the reaction mechanism and enables a novel enzyme-substrate imine to be trapped on the type I enzyme.
Asunto(s)
Ácidos Ciclohexanocarboxílicos/metabolismo , Ciclohexanonas/metabolismo , Hidroliasas/metabolismo , Isoenzimas/metabolismo , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Mycobacterium tuberculosis/enzimología , EstereoisomerismoRESUMEN
ATP-phosphoribosyltransferase (ATP-PRT) from Escherichia coli has been purified and crystals were obtained by the vapour-diffusion method using sodium tartrate as a precipitant. Dynamic light scattering was used to assess conditions for the monodispersity of the enzyme. The crystals are trigonal, space group R32, with unit-cell parameters a = b = 133.6, c= 114.1 A (at 100 K), and diffract to 2.7 A on a synchrotron X-ray source. The asymmetric unit is likely to contain one molecule, corresponding to a packing density of 2.9 A3 Da(-1). A model for the quaternary structure is proposed based on the crystallographic symmetry.
Asunto(s)
ATP Fosforribosil Transferasa/aislamiento & purificación , Escherichia coli/enzimología , ATP Fosforribosil Transferasa/química , Cristalización , Cristalografía por Rayos X , Luz , Modelos Moleculares , Conformación Proteica , Dispersión de RadiaciónRESUMEN
The structures of enzymes catalyzing the reactions in central metabolic pathways are generally well conserved as are their catalytic mechanisms. The two types of 3-dehydroquinate dehydratase (DHQase) are therefore most unusual since they are unrelated at the sequence level and they utilize completely different mechanisms to catalyze the same overall reaction. The type I enzymes catalyze a cis-dehydration of 3-dehydroquinate via a covalent imine intermediate, while the type II enzymes catalyze a trans-dehydration via an enolate intermediate. Here we report the three-dimensional structures of a representative member of each type of biosynthetic DHQase. Both enzymes function as part of the shikimate pathway, which is essential in microorganisms and plants for the biosynthesis of aromatic compounds including folate, ubiquinone and the aromatic amino acids. An explanation for the presence of two different enzymes catalyzing the same reaction is presented. The absence of the shikimate pathway in animals makes it an attractive target for antimicrobial agents. The availability of these two structures opens the way for the design of highly specific enzyme inhibitors with potential importance as selective therapeutic agents.
Asunto(s)
Hidroliasas/química , Hidroliasas/metabolismo , Mycobacterium tuberculosis/enzimología , Salmonella typhi/enzimología , Sitios de Unión , Borohidruros/metabolismo , Catálisis , Cristalización , Cristalografía por Rayos X , Dimerización , Diseño de Fármacos , Inhibidores Enzimáticos , Hidroliasas/antagonistas & inhibidores , Hidroliasas/clasificación , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Secundaria de Proteína , Ácido Shikímico/metabolismo , Relación Estructura-ActividadRESUMEN
The dodecameric type II dehydroquinases (DHQases) have an unusual quaternary structure in which four trimeric units are arranged with cubic 23 symmetry. The unfolding and refolding behaviour of the enzymes from Streptomyces coelicolor and Mycobacterium tuberculosis have been studied. Gel-permeation studies show that, at low concentrations (0.5 M) of guanidinium chloride (GdmCl), both enzymes dissociate into trimeric units, with little or no change in the secondary or tertiary structure and with a 15% loss (S. coelicolor) or a 55% increase (M. tuberculosis) in activity. At higher concentrations of GdmCl, both enzymes undergo sharp unfolding transitions over narrow ranges of the denaturant concentration, consistent with co-operative unfolding of the subunits. When the concentration of GdmCl is lowered by dilution from 6 M to 0.55 M, the enzyme from S. coelicolor refolds in an efficient manner to form trimeric units, with more than 75% regain of activity. Using a similar approach the M. tuberculosis enzyme regains less than 35% activity. From the time courses of the changes in CD, fluorescence and activity of the S. coelicolor enzyme, an outline model for the refolding of the enzyme has been proposed. The model involves a rapid refolding event in which approximately half the secondary structure is regained. A slower folding process follows within the monomer, resulting in acquisition of the full secondary structure. The major changes in fluorescence occur in a second-order process which involves the association of two folded monomers. Regain of activity is dependent on a further associative event, showing that the minimum active unit must be at least trimeric. Reassembly of the dodecameric S. coelicolor enzyme and essentially complete regain of activity can be accomplished if the denatured enzyme is dialysed extensively to remove GdmCl. These results are discussed in terms of the recently solved X-ray structures of type II DHQases from these sources.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Hidroliasas/química , Hidroliasas/metabolismo , Pliegue de Proteína , Cromatografía en Gel , Simulación por Computador , Activación Enzimática/efectos de los fármacos , Guanidina , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Conformación Proteica/efectos de los fármacos , Espectrometría de Fluorescencia , Streptomyces/enzimologíaRESUMEN
Parasites of the phylum Apicomplexa cause substantial morbidity, mortality and economic losses, and new medicines to treat them are needed urgently. The shikimate pathway is an attractive target for herbicides and antimicrobial agents because it is essential in algae, higher plants, bacteria and fungi, but absent from mammals. Here we present biochemical, genetic and chemotherapeutic evidence for the presence of enzymes of the shikimate pathway in apicomplexan parasites. In vitro growth of Toxoplasma gondii, Plasmodium falciparum (malaria) and Cryptosporidium parvum was inhibited by the herbicide glyphosate, a well-characterized inhibitor of the shikimate pathway enzyme 5-enolpyruvyl shikimate 3-phosphate synthase. This effect on T. gondii and P. falciparum was reversed by treatment with p-aminobenzoate, which suggests that the shikimate pathway supplies folate precursors for their growth. Glyphosate in combination with pyrimethamine limited T. gondii infection in mice. Four shikimate pathway enzymes were detected in extracts of T. gondii and glyphosate inhibited 5-enolpyruvyl shikimate 3-phosphate synthase activity. Genes encoding chorismate synthase, the final shikimate pathway enzyme, were cloned from T. gondii and P. falciparum. This discovery of a functional shikimate pathway in apicomplexan parasites provides several targets for the development of new antiparasite agents.
Asunto(s)
Cryptosporidium parvum/metabolismo , Plasmodium falciparum/metabolismo , Ácido Shikímico/metabolismo , Toxoplasma/metabolismo , 3-Fosfoshikimato 1-Carboxiviniltransferasa , Transferasas Alquil y Aril/antagonistas & inhibidores , Transferasas Alquil y Aril/metabolismo , Secuencia de Aminoácidos , Animales , Antiprotozoarios/farmacología , Cryptosporidium parvum/efectos de los fármacos , Cryptosporidium parvum/enzimología , Inhibidores Enzimáticos/farmacología , Glicina/análogos & derivados , Glicina/farmacología , Herbicidas/farmacología , Datos de Secuencia Molecular , Liasas de Fósforo-Oxígeno/metabolismo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Homología de Secuencia de Aminoácido , Toxoplasma/efectos de los fármacos , Toxoplasma/enzimología , GlifosatoRESUMEN
The three-dimensional structure of shikimate kinase from Erwinia chrysanthemi has been determined by multiple isomorphous replacement. Two models are presented: a high resolution 1.9 A model and a 2.6 A model which contains bound Mg-ADP. The enzyme is an alpha/beta protein consisting of a central sheet of five parallel beta-strands flanked by alpha-helices with overall topology similar to adenylate kinase. Evidence is presented that shikimate kinase undergoes major conformational changes on ligand binding. It resembles adenylate kinase in having a P-loop containing core structure and two flexible domains which undergo induced fit movement on substrate binding. The binding of Mg2+ in the active site of shikimate kinase involves direct interaction with two protein side-chains which is different from the situation found in adenylate kinase. Shikimate kinase has a readily identifiable Walker A-motif and a recognisable but modified Walker B-motif. Comparison of shikimate kinase to adenylate kinase has led to the identification of an adenine-binding motif (I/VDAXQ/NXP). Difference Fourier calculations have revealed the shikimate binding site which corresponds to the location of the AMP-binding site in adenylate kinase. A model for shikimate-binding is presented.
Asunto(s)
Dickeya chrysanthemi/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Adenosina Difosfato/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Ligandos , Magnesio/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Unión Proteica , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Type I dehydroquinase from the shikimate pathway of Escherichia coli dehydrates dehydroquinate to dehydroshikimate. pH/log Vmax profiles of the enzyme indicate the presence of a single ionizing group with a pKa of 6.2. Chemical modification experiments with diethyl pyrocarbonate have identified the conserved residue His-143 as essential for catalysis in this enzyme and the pKa for this modification is also 6.2, implying that this is the single ionizing residue in dehydroquinase that may be acting as a general base in the catalytic mechanism. Subsequent mutagenesis of this residue (Leech, A. P., James, R., Coggins, J. R., and Kleanthous, C. (1995) J. Biol. Chem. 270, 25827-25836) further suggested that His-143 may be involved in Schiff base formation/breakdown as well as being the proton abstracting general base. The importance of this residue was confirmed by recent x-ray crystallographic data showing His-143 to be at the center of a hydrogen-bonded triad, flanked by the essential Schiff base forming residue Lys-170 and Glu-86. In the present study, we have used mutagenesis and 1H and 13C NMR to assign the resonance of His-143 and probe its ionization state to define more precisely its role in the mechanism of type I dehydroquinase. Following isotopic enrichment of wild-type and H143A dehydroquinase enzymes with [2-13C]histidine, the resonance for His-143 was assigned by comparing their 1H,13C heteronuclear single quantum correlation NMR spectra. pH titrations revealed that whether in the liganded or unliganded state, His-143 does not ionize over the pH range 6-9.5 and so cannot possess a pKa of 6.2. The NMR data are consistent with this residue remaining unprotonated at pH values optimal for the activity of this enzyme (pH > 7). The role of His-143 is re-evaluated in light of these and the recent structural data, and an alternative candidate for the pKa of 6.2 is discussed.
Asunto(s)
Escherichia coli/enzimología , Histidina , Hidroliasas/química , Estructura Secundaria de Proteína , Isótopos de Carbono , Cristalografía por Rayos X , Hidroliasas/metabolismo , Hidrógeno , Enlace de Hidrógeno , Cinética , Conformación Molecular , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular/métodos , Mutación Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Bases de Schiff , Ácido Shikímico/análogos & derivados , Ácido Shikímico/química , Ácido Shikímico/metabolismoRESUMEN
From May through November 1993 and 1994, Lymnaea stagnalis appressa were collected from a stream adjacent to Cedarburg Bog, Ozaukee County, Wisconsin. Observations of cercarial shedding and subsequent dissection of snails revealed a relatively depauperate digenean component community consisting primarily of 3 common species. The most frequently occurring form was the metacercaria of Cotylurus flabelliformis. Sporocysts and cercariae of this species were rarely observed. Snails also harbored sporocysts and shed cercariae of Schistosomatium douthitti and a plagiorchid. Seasonal and yearly dynamics of the component trematode community reflect the apparent inability of sporocysts to overwinter in snails at this location. They also seem to be associated closely with snail life histories in terms of the availability of large snails, most often infected by the sporocysts of S. douthitti and the plagiorchid. Analyses of double infections revealed the occurrence of a negative interspecific interaction between the tetracotyles of C. flabelliformis and sporocysts of S. douthitti. Other combinations of trematode species did not reveal negative interactions. Competition does not seem to play a major role in the structure of the component community at this location.
Asunto(s)
Lymnaea/parasitología , Trematodos/aislamiento & purificación , Animales , Agua Dulce , Lymnaea/anatomía & histología , Prevalencia , Estaciones del Año , Factores de Tiempo , Trematodos/clasificación , Trematodos/fisiología , WisconsinRESUMEN
A simple method to identify functional amino acids in enzymes is described. This method is based on the mass spectrometric detection of molecular weight changes as the consequence of chemical modification of enzymes with group-specific reagents. Here we report the use of phenylglyoxal, trinitrobenzene sulfonic acid, tetranitromethane and diethylpyrocarbonate to identify functional amino acid residues. Precise information is obtained about the stoichiometry of reaction, and a relationship between the loss of enzyme activity and the amount of chemical modification is easily established. Modification sites are located by proteolytic digestion of the modified enzyme, followed by peptide mapping based on high-pressure liquid chromatography using an electrospray mass spectrometer as an on-line detector. In comparison with more conventional methods, protein modification is monitored directly without the need to use radioactively or spectrally labelled reagents. The methodology is limited only by the stability of the chemically modified species produced. The method has been used to characterise the active sites of several shikimate pathway enzymes, and the results obtained have been confirmed by site-directed mutagenesis and X-ray crystallography.
Asunto(s)
Enzimas/química , Espectrometría de Masas/métodos , Aminoácidos/química , Cromatografía Líquida de Alta Presión , Indicadores y Reactivos , Mapeo PeptídicoRESUMEN
Chemical cross-linking was used to study the interactions of the anti-cell-death protein Bcl2 with other proteins in the outer mitochondrial membrane. Cross-linking of mitochondrial surface proteins produced a large Bcl2-containing complex (>200 kDa), and a Bcl2-derived peptide was shown to cross-link specifically with a mitochondrial protein identified by immunoblotting as Raf-1 kinase.
Asunto(s)
Apoptosis , Reactivos de Enlaces Cruzados/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Reactivos de Enlaces Cruzados/química , Humanos , Immunoblotting , Membranas Intracelulares/química , Proteínas de la Membrana/química , Ratones , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas c-raf , Succinimidas/química , Succinimidas/metabolismo , Células Tumorales Cultivadas , Rayos UltravioletaRESUMEN
Shikimate kinase from Erwinia chrysanthemi, overexpressed in Escherichia coli has been crystallized by the vapour-diffusion method using sodium chloride as a precipitant. Mass spectrometry was used to confirm the purity of the shikimate kinase and dynamic light scattering was used to assess conditions for the monodispersity of the enzyme. The crystals are tetragonal, space group P4(1)2(1)2 or enantiomorph with cell dimensions a = b = 108.5 and c = 92.8 A (at 100 K). Native crystals diffract to better than 2.6 A on a synchrotron X-ray source. The asymmetric unit is likely to contain two molecules, corresponding to a packing density of 3.6 A(3) Da(-1).