RESUMEN

Malaria is caused by infection with protozoan parasites of the Plasmodium genus, which is part of the phylum Apicomplexa. Most organisms in this phylum contain a relic plastid called the apicoplast. The apicoplast genome is replicated by a single DNA polymerase (apPOL), which is an attractive target for anti-malarial drugs. We screened small-molecule libraries (206,504 compounds) using a fluorescence-based high-throughput DNA polymerase assay. Dose/response analysis and counter-screening identified 186 specific apPOL inhibitors. Toxicity screening against human HepaRG human cells removed 84 compounds and the remaining were subjected to parasite killing assays using chloroquine resistant P. falciparum parasites. Nine compounds were potent inhibitors of parasite growth and may serve as lead compounds in efforts to discover novel malaria drugs.

Asunto(s)

Antimaláricos , Apicoplastos , Malaria , Antimaláricos/farmacología , Apicoplastos/genética , ADN , ADN Polimerasa Dirigida por ADN , Humanos , Plasmodium falciparum , Proteínas Protozoarias/genética

RESUMEN

Glycosyltransferases (GTs) are a large family of enzymes that add sugars to a broad range of acceptor substrates, including polysaccharides, proteins and lipids, by utilizing a wide variety of donor substrates in the form of activated sugars. Individual GTs have generally been considered to exhibit a high level of substrate specificity, but this has not been thoroughly investigated across the extremely large set of GTs. Here we investigate xyloglucan xylosyltransferase 1 (XXT1), a GT involved in the synthesis of the plant cell wall polysaccharide, xyloglucan. Xyloglucan has a glucan backbone, with initial side chain substitutions exclusively composed of xylose from uridine diphosphate (UDP)-xylose. While this conserved substitution pattern suggests a high substrate specificity for XXT1, our in vitro kinetic studies elucidate a more complex set of behavior. Kinetic studies demonstrate comparable kcat values for reactions with UDP-xylose and UDP-glucose, while reactions with UDP-arabinose and UDP-galactose are over 10-fold slower. Using kcat/KM as a measure of efficiency, UDP-xylose is 8-fold more efficient as a substrate than the next best alternative, UDP-glucose. To the best of our knowledge, we are the first to demonstrate that not all plant XXTs are highly substrate specific and some do show significant promiscuity in their in vitro reactions. Kinetic parameters alone likely do not explain the high substrate selectivity in planta, suggesting that there are additional control mechanisms operating during polysaccharide biosynthesis. Improved understanding of substrate specificity of the GTs will aid in protein engineering, development of diagnostic tools, and understanding of biological systems.

Asunto(s)

Glucanos/biosíntesis , Pentosiltransferasa/genética , Proteínas de Plantas/genética , Plantas/enzimología , Glucanos/genética , Cinética , Pentosiltransferasa/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Especificidad por Sustrato

RESUMEN

The plant cell wall is primarily a polysaccharide mesh of the most abundant biopolymers on earth. Although one of the richest sources of biorenewable materials, the biosynthesis of the plant polysaccharides is poorly understood. Structures of many essential plant glycosyltransferases are unknown and suitable substrates are often unavailable for in vitro analysis. The dearth of such information impedes the development of plants better suited for industrial applications. Presented here are structures of Arabidopsis xyloglucan xylosyltransferase 1 (XXT1) without ligands and in complexes with UDP and cellohexaose. XXT1 initiates side-chain extensions from a linear glucan polymer by transferring the xylosyl group from UDP-xylose during xyloglucan biosynthesis. XXT1, a homodimer and member of the GT-A fold family of glycosyltransferases, binds UDP analogously to other GT-A fold enzymes. Structures here and the properties of mutant XXT1s are consistent with a SNi-like catalytic mechanism. Distinct from other systems is the recognition of cellohexaose by way of an extended cleft. The XXT1 dimer alone cannot produce xylosylation patterns observed for native xyloglucans because of steric constraints imposed by the acceptor binding cleft. Homology modeling of XXT2 and XXT5, the other two xylosyltransferases involved in xyloglucan biosynthesis, reveals a structurally altered cleft in XXT5 that could accommodate a partially xylosylated glucan chain produced by XXT1 and/or XXT2. An assembly of the three XXTs can produce the xylosylation patterns of native xyloglucans, suggesting the involvement of an organized multienzyme complex in the xyloglucan biosynthesis.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/ultraestructura , Pentosiltransferasa/metabolismo , Pentosiltransferasa/ultraestructura , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Pared Celular/metabolismo , Cristalografía por Rayos X/métodos , Glucanos/genética , Glucanos/metabolismo , Modelos Biológicos , Pentosiltransferasa/genética , Xilanos/genética , Xilanos/metabolismo , UDP Xilosa Proteína Xilosiltransferasa

RESUMEN

Plasmodium falciparum, the primary cause of malaria, contains a non-photosynthetic plastid called the apicoplast. The apicoplast exists in most members of the phylum Apicomplexa and has its own genome along with organelle-specific enzymes for its replication. The only DNA polymerase found in the apicoplast (apPOL) was putatively acquired through horizontal gene transfer from a bacteriophage and is classified as an atypical A-family polymerase. Here, we present its crystal structure at a resolution of 2.9Å. P. falciparum apPOL, the first structural representative of a plastidic A-family polymerase, diverges from typical A-family members in two of three previously identified signature motifs and in a region not implicated by sequence. Moreover, apPOL has an additional N-terminal subdomain, the absence of which severely diminishes its 3' to 5' exonuclease activity. A compound known to be toxic to Plasmodium is a potent inhibitor of apPOL, suggesting that apPOL is a viable drug target. The structure provides new insights into the structural diversity of A-family polymerases and may facilitate structurally guided antimalarial drug design.

Asunto(s)

Apicoplastos/enzimología , ADN Polimerasa Dirigida por ADN/química , Plasmodium falciparum/enzimología , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica , Dominios Proteicos

RESUMEN

Infection by the parasite Plasmodium falciparum is the leading cause of malaria in humans. The parasite has a unique and essential plastid-like organelle called the apicoplast. The apicoplast contains a genome that undergoes replication and repair through the action of a replicative polymerase (apPOL). apPOL has no direct orthologs in mammalian polymerases and is therefore an attractive antimalarial drug target. No structural information exists for apPOL, and the Klenow fragment of Escherichia coli DNA polymerase I, which is its closest structural homolog, shares only 28% sequence identity. Here, conditions for the crystallization of and preliminary X-ray diffraction data from crystals of P. falciparum apPOL are reported. Data complete to 3.5 Šresolution were collected from a single crystal (2 × 2 × 5 µm) using a 5 µm beam. The space group P6522 (unit-cell parameters a = b = 141.8, c = 149.7 Å, α = ß = 90, γ = 120°) was confirmed by molecular replacement. Refinement is in progress.

Asunto(s)

Apicoplastos/enzimología , ADN Polimerasa I/química , Plasmodium falciparum/enzimología , Proteínas Protozoarias/química , Cristalización , Cristalografía por Rayos X

RESUMEN

The effects of AMP and fructose 2,6-bisphosphate (Fru-2,6-P2) on porcine fructose-1,6-bisphosphatase (pFBPase) and Escherichia coli FBPase (eFBPase) differ in three respects. AMP/Fru-2,6-P2 synergism in pFBPase is absent in eFBPase. Fru-2,6-P2 induces a 13° subunit pair rotation in pFBPase but no rotation in eFBPase. Hydrophilic side chains in eFBPase occupy what otherwise would be a central aqueous cavity observed in pFBPase. Explored here is the linkage of AMP/Fru-2,6-P2 synergism to the central cavity and the evolution of synergism in FBPases. The single mutation Ser(45) → His substantially fills the central cavity of pFBPase, and the triple mutation Ser(45) → His, Thr(46) → Arg, and Leu(186) → Tyr replaces porcine with E. coli type side chains. Both single and triple mutations significantly reduce synergism while retaining other wild-type kinetic properties. Similar to the effect of Fru-2,6-P2 on eFBPase, the triple mutant of pFBPase with bound Fru-2,6-P2 exhibits only a 2° subunit pair rotation as opposed to the 13° rotation exhibited by the Fru-2,6-P2 complex of wild-type pFBPase. The side chain at position 45 is small in all available eukaryotic FBPases but large and hydrophilic in bacterial FBPases, similar to eFBPase. Sequence information indicates the likelihood of synergism in the FBPase from Leptospira interrogans (lFBPase), and indeed recombinant lFBPase exhibits AMP/Fru-2,6-P2 synergism. Unexpectedly, however, AMP also enhances Fru-6-P binding to lFBPase. Taken together, these observations suggest the evolution of AMP/Fru-2,6-P2 synergism in eukaryotic FBPases from an ancestral FBPase having a central aqueous cavity and exhibiting synergistic feedback inhibition by AMP and Fru-6-P.

Asunto(s)

Adenosina Monofosfato/metabolismo , Fructosa-Bifosfatasa/metabolismo , Fructosadifosfatos/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Evolución Molecular , Fructosa-Bifosfatasa/química , Fructosa-Bifosfatasa/genética , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Porcinos

RESUMEN

AMP triggers a 15° subunit-pair rotation in fructose-1,6-bisphosphatase (FBPase) from its active R state to its inactive T state. During this transition, a catalytically essential loop (residues 50-72) leaves its active (engaged) conformation. Here, the structures of Ile(10) → Asp FBPase and molecular dynamic simulations reveal factors responsible for loop displacement. The AMP/Mg(2+) and AMP/Zn(2+) complexes of Asp(10) FBPase are in intermediate quaternary conformations (completing 12° of the subunit-pair rotation), but the complex with Zn(2+) provides the first instance of an engaged loop in a near-T quaternary state. The 12° subunit-pair rotation generates close contacts involving the hinges (residues 50-57) and hairpin turns (residues 58-72) of the engaged loops. Additional subunit-pair rotation toward the T state would make such contacts unfavorable, presumably causing displacement of the loop. Targeted molecular dynamics simulations reveal no steric barriers to subunit-pair rotations of up to 14° followed by the displacement of the loop from the active site. Principal component analysis reveals high-amplitude motions that exacerbate steric clashes of engaged loops in the near-T state. The results of the simulations and crystal structures are in agreement: subunit-pair rotations just short of the canonical T state coupled with high-amplitude modes sterically displace the dynamic loop from the active site.

Asunto(s)

Fructosa-Bifosfatasa/química , Sus scrofa/genética , Adenosina Monofosfato/química , Adenosina Monofosfato/metabolismo , Animales , Sitios de Unión , Catálisis , Dominio Catalítico , Activación Enzimática , Fructosa-Bifosfatasa/metabolismo , Simulación de Dinámica Molecular , Estructura Secundaria de Proteína , Sus scrofa/metabolismo

RESUMEN

The complexity of terpenoid natural products has drawn significant interest, particularly since their common (poly)isoprenyl origins were discovered. Notably, much of this complexity is derived from the highly variable cyclized and/or rearranged nature of the observed hydrocarbon skeletal structures. Indeed, at least in some cases it is difficult to immediately recognize their derivation from poly-isoprenyl precursors. Nevertheless, these diverse structures are formed by sequential elongation to acyclic precursors, most often with subsequent cyclization and/or rearrangement. Strikingly, the reactions used to assemble and diversify terpenoid backbones share a common carbocationic driven mechanism, although the means by which the initial carbocation is generated does vary. High-resolution crystal structures have been obtained for at least representative examples from each of the various types of enzymes involved in producing terpenoid hydrocarbon backbones. However, while this has certainly led to some insights into the enzymatic structure-function relationships underlying the elongation and simpler cyclization reactions, our understanding of the more complex cyclization and/or rearrangement reactions remains limited. Accordingly, selected examples are discussed here to demonstrate our current understanding, its limits, and potential ways forward.

Asunto(s)

Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/metabolismo , Terpenos/química , Terpenos/metabolismo , Catálisis , Estructura Molecular , Conformación Proteica , Relación Estructura-Actividad

RESUMEN

The crystal structure of the interleukin-2 tyrosine kinase Src homology domain (Itk SH2) is described and it is found that unlike in studies of this domain using NMR spectroscopy, cis-trans-prolyl isomerization is not readily detected in the crystal structure. Based on similarities between the Itk SH2 crystal form and the cis form of the Itk SH2 NMR structure, it is concluded that it is likely that the prolyl imide bond at least in part adopts the cis conformation in the crystal form. However, the lack of high-resolution data and the dynamic nature of the proline-containing loop mean that the precise imide-bond conformation cannot be determined and prolyl cis-trans isomerization in the crystal cannot be ruled out. Given the preponderance of structures that have been solved by X-ray crystallography in the Protein Data Bank, this result supports the notion that prolyl isomerization in folded proteins has been underestimated among known structures. Interestingly, while the precise status of the proline residue is ambiguous, Itk SH2 crystallizes as a domain-swapped dimer. The domain-swapped structure of Itk SH2 is similar to the domain-swapped SH2 domains of Grb2 and Nck, with domain swapping occurring at the ß-meander region of all three SH2 domains. Thus, for Itk SH2 structural analysis by NMR spectroscopy and X-ray crystallography revealed very different structural features: proline isomerization versus domain-swapped dimerization, respectively.

Asunto(s)

Proteínas Tirosina Quinasas/química , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Estructura Cuaternaria de Proteína , Proteínas Tirosina Quinasas/genética

RESUMEN

Abietadiene synthase from Abies grandis (AgAS) is a model system for diterpene synthase activity, catalyzing class I (ionization-initiated) and class II (protonation-initiated) cyclization reactions. Reported here is the crystal structure of AgAS at 2.3 Å resolution and molecular dynamics simulations of that structure with and without active site ligands. AgAS has three domains (α, ß, and γ). The class I active site is within the C-terminal α domain, and the class II active site is between the N-terminal γ and ß domains. The domain organization resembles that of monofunctional diterpene synthases and is consistent with proposed evolutionary origins of terpene synthases. Molecular dynamics simulations were carried out to determine the effect of substrate binding on enzymatic structure. Although such studies of the class I active site do lead to an enclosed substrate-Mg(2+) complex similar to that observed in crystal structures of related plant enzymes, it does not enforce a single substrate conformation consistent with the known product stereochemistry. Simulations of the class II active site were more informative, with observation of a well ordered external loop migration. This "loop-in" conformation not only limits solvent access but also greatly increases the number of conformational states accessible to the substrate while destabilizing the nonproductive substrate conformation present in the "loop-out" conformation. Moreover, these conformational changes at the class II active site drive the substrate toward the proposed transition state. Docked substrate complexes were further assessed with regard to the effects of site-directed mutations on class I and II activities.

Asunto(s)

Abies/enzimología , Diterpenos/química , Diterpenos/metabolismo , Isomerasas/química , Isomerasas/metabolismo , Sitios de Unión , Dominio Catalítico , Simulación por Computador , Cristalografía por Rayos X , Ciclización , Isomerasas/genética , Modelos Químicos , Mutagénesis Sitio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína

RESUMEN

Proline is a unique amino acid owing to the relatively small energy difference between the cis and trans conformations of its peptide bond. The X-Pro imide bond readily undergoes cis-trans isomerization in the context of short peptides as well as some proteins. However, the direct detection of cis-trans proline isomerization in folded proteins is technically challenging. NMR spectroscopy is well suited to the direct detection of proline isomerization in folded proteins. It is less clear how well X-ray crystallography can reveal this conformational exchange event in folded proteins. Conformational heterogeneity owing to cis-trans proline isomerization in the Src homology 2 (SH2) domain of the IL-2-inducible T-cell kinase (ITK) has been extensively characterized by NMR. Using the ITK SH2 domain as a test system, an attempt was made to determine whether proline isomerization could be detected in a crystal structure of the ITK SH2 domain. As a first step towards this goal, the purification, crystallization and preliminary characterization of the ITK SH2 domain are described.

Asunto(s)

Proteínas Tirosina Quinasas/química , Dominios Homologos src , Animales , Cristalización , Cristalografía por Rayos X , Ratones , Conformación Molecular , Péptidos/metabolismo , Prolina/química , Prolina/metabolismo , Difracción de Rayos X

RESUMEN

VP35 is one of seven structural proteins encoded by the Ebola viral genome and mediates viral replication, nucleocapsid formation and host immune suppression. The C-terminal interferon inhibitory domain (IID) of VP35 is critical for dsRNA binding and interferon inhibition. The wild-type VP35 IID structure revealed several conserved residues that are important for dsRNA binding and interferon antagonism. Here, the expression, purification and crystallization of recombinant Zaire Ebola VP35 IID mutants R312A, K319A/R322A and K339A in space groups P6(1)22, P2(1)2(1)2(1) and P2(1), respectively, are described. Diffraction data were collected using synchrotron sources at the Advanced Light Source and the Advanced Photon Source.

Asunto(s)

Ebolavirus/química , Interferones/antagonistas & inhibidores , Mutación , Proteínas Reguladoras y Accesorias Virales/química , Secuencia de Aminoácidos , Secuencia Conservada , Cristalización , Cristalografía por Rayos X , Ebolavirus/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Proteínas Reguladoras y Accesorias Virales/genética

RESUMEN

Ebola viral protein 35 (VP35), encoded by the highly pathogenic Ebola virus, facilitates host immune evasion by antagonizing antiviral signaling pathways, including those initiated by RIG-I-like receptors. Here we report the crystal structure of the Ebola VP35 interferon inhibitory domain (IID) bound to short double-stranded RNA (dsRNA), which together with in vivo results reveals how VP35-dsRNA interactions contribute to immune evasion. Conserved basic residues in VP35 IID recognize the dsRNA backbone, whereas the dsRNA blunt ends are 'end-capped' by a pocket of hydrophobic residues that mimic RIG-I-like receptor recognition of blunt-end dsRNA. Residues critical for RNA binding are also important for interferon inhibition in vivo but not for viral polymerase cofactor function of VP35. These results suggest that simultaneous recognition of dsRNA backbone and blunt ends provides a mechanism by which Ebola VP35 antagonizes host dsRNA sensors and immune responses.

Asunto(s)

Interferones/antagonistas & inhibidores , ARN Bicatenario/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/inmunología , Ebolavirus/química , Ebolavirus/inmunología , Evasión Inmune , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Receptores Inmunológicos

RESUMEN

A gene encoding a glycoside hydrolase family 44 (GH44) protein from Clostridium acetobutylicum ATCC 824 was synthesized and transformed into Escherichia coli. The previously uncharacterized protein was expressed with a C-terminal His tag and purified by nickel-nitrilotriacetic acid affinity chromatography. Crystallization and X-ray diffraction to a 2.2-A resolution revealed a triose phosphate isomerase (TIM) barrel-like structure with additional Greek key and beta-sandwich folds, similar to other GH44 crystal structures. The enzyme hydrolyzes cellotetraose and larger cellooligosaccharides, yielding an unbalanced product distribution, including some glucose. It attacks carboxymethylcellulose and xylan at approximately the same rates. Its activity on carboxymethylcellulose is much higher than that of the isolated C. acetobutylicum cellulosome. It also extensively converts lichenan to oligosaccharides of intermediate size and attacks Avicel to a limited extent. The enzyme has an optimal temperature in a 10-min assay of 55 degrees C and an optimal pH of 5.0.

Asunto(s)

Celulasa/química , Celulasa/metabolismo , Clostridium acetobutylicum/enzimología , Carboximetilcelulosa de Sodio/metabolismo , Celulasa/genética , Celulasa/aislamiento & purificación , Celulosa/análogos & derivados , Celulosa/metabolismo , Clostridium acetobutylicum/genética , Cristalización , Cristalografía por Rayos X , Estabilidad de Enzimas , Escherichia coli/genética , Expresión Génica , Glucanos/metabolismo , Glucosa/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Oligosacáridos/metabolismo , Filogenia , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Temperatura , Tetrosas/metabolismo , Transformación Genética , Xilanos/metabolismo

RESUMEN

Ebola VP35 is a multifunctional protein that is important for host immune suppression and pathogenesis. VP35 contains an N-terminal oligomerization domain and a C-terminal interferon inhibitory domain (IID). Mutations within the VP35 IID result in loss of host immune suppression. Here, efforts to crystallize recombinantly overexpressed VP35 IID that was purified from Escherichia coli are described. Native and selenomethionine-labeled crystals belonging to the orthorhombic space group P2(1)2(1)2(1) were obtained by the hanging-drop vapor-diffusion method and diffraction data were collected at the ALS synchrotron.

Asunto(s)

Ebolavirus/química , Ebolavirus/patogenicidad , Regulación Viral de la Expresión Génica/fisiología , Interferones/antagonistas & inhibidores , Nucleoproteínas/química , Nucleoproteínas/fisiología , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/fisiología , Difracción de Rayos X , Cristalización , Interferones/química , Proteínas de la Nucleocápside , Nucleoproteínas/biosíntesis , Estructura Terciaria de Proteína/fisiología , Proteínas del Núcleo Viral/biosíntesis , Proteínas Reguladoras y Accesorias Virales/biosíntesis , Proteínas Reguladoras y Accesorias Virales/química , Proteínas Reguladoras y Accesorias Virales/aislamiento & purificación , Virulencia

RESUMEN

Ebola viruses (EBOVs) cause rare but highly fatal outbreaks of viral hemorrhagic fever in humans, and approved treatments for these infections are currently lacking. The Ebola VP35 protein is multifunctional, acting as a component of the viral RNA polymerase complex, a viral assembly factor, and an inhibitor of host interferon (IFN) production. Mutation of select basic residues within the C-terminal half of VP35 abrogates its dsRNA-binding activity, impairs VP35-mediated IFN antagonism, and attenuates EBOV growth in vitro and in vivo. Because VP35 contributes to viral escape from host innate immunity and is required for EBOV virulence, understanding the structural basis for VP35 dsRNA binding, which correlates with suppression of IFN activity, is of high importance. Here, we report the structure of the C-terminal VP35 IFN inhibitory domain (IID) solved to a resolution of 1.4 A and show that VP35 IID forms a unique fold. In the structure, we identify 2 basic residue clusters, one of which is important for dsRNA binding. The dsRNA binding cluster is centered on Arg-312, a highly conserved residue required for IFN inhibition. Mutation of residues within this cluster significantly changes the surface electrostatic potential and diminishes dsRNA binding activity. The high-resolution structure and the identification of the conserved dsRNA binding residue cluster provide opportunities for antiviral therapeutic design. Our results suggest a structure-based model for dsRNA-mediated innate immune antagonism by Ebola VP35 and other similarly constructed viral antagonists.

Asunto(s)

Ebolavirus/química , Proteínas Reguladoras y Accesorias Virales/química , Sitios de Unión , Cristalografía por Rayos X , Interferones/antagonistas & inhibidores , Conformación Proteica , ARN Bicatenario/metabolismo , ARN Viral/metabolismo , Proteínas de Unión al ARN/química , Proteínas Virales

RESUMEN

Fructose-1,6-bisphosphatase (FBPase) operates at a control point in mammalian gluconeogenesis, being inhibited synergistically by fructose 2,6-bisphosphate (Fru-2,6-P(2)) and AMP. AMP and Fru-2,6-P(2) bind to allosteric and active sites, respectively, but the mechanism responsible for AMP/Fru-2,6-P(2) synergy is unclear. Demonstrated here for the first time is a global conformational change in porcine FBPase induced by Fru-2,6-P(2) in the absence of AMP. The Fru-2,6-P(2) complex exhibits a subunit pair rotation of 13 degrees from the R-state (compared with the 15 degrees rotation of the T-state AMP complex) with active site loops in the disengaged conformation. A three-state thermodynamic model in which Fru-2,6-P(2) drives a conformational change to a T-like intermediate state can account for AMP/Fru-2,6-P(2) synergism in mammalian FBPases. AMP and Fru-2,6-P(2) are not synergistic inhibitors of the Type I FBPase from Escherichia coli, and consistent with that model, the complex of E. coli FBPase with Fru-2,6-P(2) remains in the R-state with dynamic loops in the engaged conformation. Evidently in porcine FBPase, the actions of AMP at the allosteric site and Fru-2,6-P(2) at the active site displace engaged dynamic loops by distinct mechanisms, resulting in similar quaternary end-states. Conceivably, Type I FBPases from all eukaryotes may undergo similar global conformational changes in response to Fru-2,6-P(2) ligation.

Asunto(s)

Adenosina Monofosfato/química , Escherichia coli/enzimología , Fructosa-Bifosfatasa/antagonistas & inhibidores , Fructosa-Bifosfatasa/química , Fructosadifosfatos/química , Modelos Químicos , Porcinos/metabolismo , Adenosina Monofosfato/agonistas , Sitio Alostérico/fisiología , Animales , Fructosadifosfatos/agonistas , Gluconeogénesis/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Termodinámica

RESUMEN

Allosteric activation of fructose-1,6-bisphosphatase (FBPase) from Escherichia coli by phosphoenolpyruvate implies rapid feed-forward activation of gluconeogenesis in heterotrophic bacteria. But how do such bacteria rapidly down-regulate an activated FBPase in order to avoid futile cycling? Demonstrated here is the allosteric inhibition of E. coli FBPase by glucose 6-phosphate (Glc-6-P), the first metabolite produced upon glucose transport into the cell. FBPase undergoes a quaternary transition from the canonical R-state to a T-like state in response to Glc-6-P and AMP ligation. By displacing Phe(15), AMP binds to an allosteric site comparable with that of mammalian FBPase. Relative movements in helices H1 and H2 perturb allosteric activator sites for phosphoenolpyruvate. Glc-6-P binds to allosteric sites heretofore not observed in previous structures, perturbing subunits that in pairs form complete active sites of FBPase. Glc-6-P and AMP are synergistic inhibitors of E. coli FBPase, placing AMP/Glc-6-P inhibition in bacteria as a possible evolutionary predecessor to AMP/fructose 2,6-bisphosphate inhibition in mammalian FBPases. With no exceptions, signature residues of allosteric activation appear in bacterial sequences along with key residues of the Glc-6-P site. FBPases in such organisms may be components of metabolic switches that allow rapid changeover between gluconeogenesis and glycolysis in response to nutrient availability.

Asunto(s)

Escherichia coli/enzimología , Fructosa-Bifosfatasa/antagonistas & inhibidores , Fructosa-Bifosfatasa/química , Adenosina Monofosfato/química , Sitio Alostérico , Sitios de Unión , Relación Dosis-Respuesta a Droga , Escherichia coli/genética , Gluconeogénesis , Glucosa-6-Fosfato/química , Cinética , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Estereoisomerismo , Factores de Tiempo

RESUMEN

The enteric bacterium Escherichia coli requires fructose-1,6-bisphosphatase (FBPase) for growth on gluconeogenic carbon sources. Constitutive expression of FBPase and fructose-6-phosphate-1-kinase coupled with the absence of futile cycling implies an undetermined mechanism of coordinate regulation involving both enzymes. Tricarboxylic acids and phosphorylated three-carbon carboxylic acids, all intermediates of glycolysis and the tricarboxylic acid cycle, are shown here to activate E. coli FBPase. The two most potent activators, phosphoenolpyruvate and citrate, bind to the sulfate anion site, revealed previously in the first crystal structure of the E. coli enzyme. Tetramers ligated with either phosphoenolpyruvate or citrate, in contrast to the sulfate-bound structure, are in the canonical R-state of porcine FBPase but nevertheless retain sterically blocked AMP pockets. At physiologically relevant concentrations, phosphoenolpyruvate and citrate stabilize an active tetramer over a less active enzyme form of mass comparable with that of a dimer. The above implies the conservation of the R-state through evolution. FBPases of heterotrophic organisms of distantly related phylogenetic groups retain residues of the allosteric activator site and in those instances where data are available exhibit activation by phosphoenolpyruvate. Findings here unify disparate observations regarding bacterial FBPases, implicating a mechanism of feed-forward activation in bacterial central metabolism.

Asunto(s)

Escherichia coli/enzimología , Fructosa-Bifosfatasa/química , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Sitio Alostérico , Animales , Ácidos Carboxílicos/química , Ácido Cítrico/química , Cristalografía por Rayos X , Dimerización , Fructosa-Bifosfatasa/fisiología , Cinética , Fosfoenolpiruvato/química , Fosforilación , Conformación Proteica , Porcinos

RESUMEN

Adenylosuccinate synthetase catalyzes the first committed step in the de novo biosynthesis of AMP, coupling L-aspartate and IMP to form adenylosuccinate. Km values of IMP and 2'-deoxy-IMP are nearly identical with each substrate supporting comparable maximal velocities. Nonetheless, the Km value for L-aspartate and the Ki value for hadacidin (a competitive inhibitor with respect to L-aspartate) are 29-57-fold lower in the presence of IMP than in the presence of 2'-deoxy-IMP. Crystal structures of the synthetase ligated with hadacidin, GDP, and either 6-phosphoryl-IMP or 2'-deoxy-6-phosphoryl-IMP are identical except for the presence of a cavity normally occupied by the 2'-hydroxyl group of IMP. In the presence of 6-phosphoryl-IMP and GDP (hadacidin absent), the L-aspartate pocket can retain its fully ligated conformation, forming hydrogen bonds between the 2'-hydroxyl group of IMP and sequence-invariant residues. In the presence of 2'-deoxy-6-phosphoryl-IMP and GDP, however, the L-aspartate pocket is poorly ordered. The absence of the 2'-hydroxyl group of the deoxyribonucleotide may destabilize binding of the ligand to the L-aspartate pocket by disrupting hydrogen bonds that maintain a favorable protein conformation and by the introduction of a cavity into the fully ligated active site. At an approximate energy cost of 2.2 kcal/mol, the unfavorable thermodynamics of cavity formation may be the major factor in destabilizing ligands at the L-aspartate pocket.

Asunto(s)

Adenilosuccinato Sintasa/metabolismo , Adenilosuccinato Sintasa/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Desoxirribonucleótidos/metabolismo , Electrones , Escherichia coli/enzimología , Inosina Monofosfato/metabolismo , Cinética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Músculos/enzimología , Especificidad por Sustrato