RESUMEN
In a continuing effort to understand reaction mechanisms of terpene synthases catalyzing initial anti-Markovnikov cyclization reactions, we solved the X-ray crystal structure of (+)-caryolan-1-ol synthase (CS) from Streptomyces griseus , with and without an inactive analog of the FPP substrate, 2-fluorofarnesyl diphosphate (2FFPP), bound in the active site of the enzyme. The CS-2FFPP complex was solved to 2.65 Å resolution and showed the ligand in a linear, elongated orientation, incapable of undergoing the initial cyclization event to form a bond between carbons C1 and C11. Intriguingly, the apo CS structure (2.2 Å) also had electron density in the active site, in this case density that was well fit with a curled-up tetraethylene glycol molecule presumably recruited from the crystallization medium. The density was also well fit by a molecule of farnesene suggesting that the structure may mimic an intermediate along the reaction coordinate. The curled-up conformation of tetraethylene glycol was accompanied by dramatic rotamer shifts among active-site residues. Most notably, W56 was observed to undergo a 90° rotation between the 2FFPP complex and apo-enzyme structures, suggesting that it contributes to steric interactions that help curl the tetraethylene glycol molecule in the active site, and by extension perhaps also a derivative of the FPP substrate in the normal course of the cyclization reaction. In support of this proposal, the CS W56L variant lost the ability to cyclize the FPP substrate and produced only the linear terpene products farnesol and α- and ß-farnesene.
RESUMEN
As sessile organisms, plants evolved elaborate metabolic systems that produce a plethora of specialized metabolites as a means to survive challenging terrestrial environments. Decades of research have revealed the genetic and biochemical basis for a multitude of plant specialized metabolic pathways. Nevertheless, knowledge is still limited concerning the selective advantages provided by individual and collective specialized metabolites to the reproductive success of diverse host plants. Here we review the biological functions conferred by various classes of plant specialized metabolites in the context of the interaction of plants with their surrounding environment. To achieve optimal multifunctionality of diverse specialized metabolic processes, plants use various adaptive mechanisms at subcellular, cellular, tissue, organ and interspecies levels. Understanding these mechanisms and the evolutionary trajectories underlying their occurrence in nature will ultimately enable efficient bioengineering of desirable metabolic traits in chassis organisms.
Asunto(s)
Adaptación Fisiológica/genética , Evolución Biológica , Epigénesis Genética/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Plantas/metabolismoRESUMEN
Aggregates of α-synuclein contribute to the etiology of Parkinson's Disease. Protein disulfide isomerase (PDI), a chaperone and oxidoreductase, blocks the aggregation of α-synuclein. An S-nitrosylated form of PDI that cannot function as a chaperone is associated with elevated levels of aggregated α-synuclein and is found in brains afflicted with Parkinson's Disease. The protective role of PDI in Parkinson's Disease and other neurodegenerative disorders is linked to its chaperone function, yet the mechanism of neuroprotection remains unclear. Using Thioflavin-T fluorescence and transmission electron microscopy, we show here for the first time that PDI can break down nascent fibrils of α-synuclein. Mature fibrils were not affected by PDI. Another PDI family member, ERp57, could prevent but not reverse α-synuclein aggregation. The disaggregase activity of PDI was effective at a 1:50 molar ratio of PDI:α-synuclein and was blocked by S-nitrosylation. PDI could not reverse the aggregation of malate dehydrogenase, which indicated its disaggregase activity does not operate on all substrates. These findings establish a previously unrecognized disaggregase property of PDI that could underlie its neuroprotective function.
RESUMEN
Most terpene synthase reactions follow Markovnikov rules for formation of high-energy carbenium ion intermediates. However, there are notable exceptions. For example, pentalenene synthase (PS) undergoes an initial anti-Markovnikov cyclization reaction followed by a 1,2-hydride shift to form an intermediate humulyl cation with positive charge on the secondary carbon C9 atom of the farnesyl diphosphate substrate. The mechanism by which these enzymes stabilize and guide the regioselectivity of secondary carbocations has not heretofore been elucidated. In an effort to better understand these reactions, we grew crystals of apo-PS, soaked them with the nonreactive substrate analogue 12,13-difluorofarnesyl diphosphate, and determined the X-ray structure of the resulting complex at 2.2 Å resolution. The most striking feature of the active site structure is that C9 is perfectly positioned to make a C-H···π interaction with the side chain benzene ring of residue F76; this would enhance hyperconjugation to stabilize a developing cation at C10 and thus support the anti-Markovnikov regioselectivity of the cyclization. The benzene ring is also positioned to catalyze the migration of H to C10 and stabilize a C9 carbocation. On the opposite face of C9, further cation stabilization is possible via interactions with the main chain carbonyl of I177 and the neighboring intramolecular C6âC7 bond. Mutagenesis experiments also support a role for residue 76 in these interactions, but most interesting is the F76W mutant, whose crystal structure clearly shows C9 and C10 centered above the fused benzene and pyrrole rings of the indole side chain, respectively, such that a carbocation at either position could be stabilized in this complex, and two anti-Markovnikov products, pentalenene and humulene, are formed. Finally, we show that there is a rough correlation (although not absolute) of an aromatic side chain (F or Y) at position 76 in related terpene synthases from Streptomyces that catalyze similar anti-Markovnikov addition reactions.
Asunto(s)
Liasas Intramoleculares/química , Liasas Intramoleculares/metabolismo , Streptomyces/enzimología , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/metabolismo , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Ciclización , Ciclopentanos/química , Ciclopentanos/metabolismo , Modelos Moleculares , Conformación ProteicaRESUMEN
Linalyl diphosphate (LPP) is the postulated intermediate in the enzymatic cyclization of monoterpenes catalyzed by terpene synthases. LPP is considered an obligate intermediate due to the conformationally restrictive trans-C2-C3 double bond of the substrate, geranyl diphosphate (GPP), which precludes the proper positioning of carbons C1 and C6 to enable cyclization. However, because of the complexity of potential carbocation-mediated rearrangements in these enzymatic reactions, it has proven difficult to directly demonstrate the formation of LPP despite significant efforts. Here we synthesized a fluorinated substrate analog, 8,9-difluorogeranyl diphosphate (DFGPP), which is designed to allow initial ionization/isomerization and form the fluorinated equivalent of LPP (DFLPP) while preventing the subsequent ionization/cyclization to produce the α-terpinyl cation. Steady-state kinetic studies with the model enzyme (+)-limonene synthase (LS) under catalytic conditions show that the cyclization of DFGPP is completely blocked and a single linear product, difluoromyrcene, is produced. When crystals of apo-LS are soaked with DFGPP under conditions limiting turnover of the enzyme, we show, using X-ray crystallography, that DFLPP is produced in the enzyme active site and trapped in the crystals. Clear electron density is observed in the active site of the enzyme, but it cannot be appropriately fit with a model for the DFGPP substrate analog, whereas it can accommodate an extended conformation of DFLPP. This result supports the current model for monoterpene cyclization by providing direct evidence of LPP as an intermediate.
Asunto(s)
Monoterpenos Acíclicos/química , Difosfatos/química , Diterpenos/química , Inhibidores Enzimáticos/química , Liasas Intramoleculares/antagonistas & inhibidores , Fosfatos de Poliisoprenilo/química , Dominio Catalítico , Citrus sinensis/enzimología , Cristalografía por Rayos X , Difosfatos/síntesis química , Diterpenos/síntesis química , Pruebas de Enzimas , Inhibidores Enzimáticos/síntesis química , Liasas Intramoleculares/químicaRESUMEN
The amyloid ß (Aß) peptide and its shorter variants, including a highly cytotoxic Aß25-35 peptide, exert their neurotoxic effect during Alzheimer's disease by various mechanisms, including cellular membrane permeabilization. The intrinsic polymorphism of Aß has prevented the identification of the molecular basis of Aß pore formation by direct structural methods, and computational studies have led to highly divergent pore models. Here, we have employed a set of biophysical techniques to directly monitor Ca2+-transporting Aß25-35 pores in lipid membranes, to quantitatively characterize pore formation, and to identify the key structural features of the pore. Moreover, the effect of membrane cholesterol on pore formation and the structure of Aß25-35 has been elucidated. The data suggest that the membrane-embedded peptide forms 6- or 8-stranded ß-barrel like structures. The 8-stranded barrels may conduct Ca2+ ions through an inner cavity, whereas the tightly packed 6-stranded barrels need to assemble into supramolecular structures to form a central pore. Cholesterol affects Aß25-35 pore formation by a dual mechanism, i.e., by direct interaction with the peptide and by affecting membrane structure. Collectively, our data illuminate the molecular basis of Aß membrane pore formation, which should advance both basic and clinical research on Alzheimer's disease and membrane-associated pathologies in general.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Fragmentos de Péptidos/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Humanos , Simulación de Dinámica Molecular , Fragmentos de Péptidos/química , Multimerización de Proteína/genética , Multimerización de Proteína/fisiologíaRESUMEN
Amyloid ß (Aß) peptide plays a major role in Alzheimer's disease (AD) and occurs in multiple forms, including pyroglutamylated Aß (AßpE). Identification and characterization of the most cytotoxic Aß species is necessary for advancement in AD diagnostics and therapeutics. While in brain tissue multiple Aß species act in combination, structure/toxicity studies and immunotherapy trials have been focused on individual forms of Aß. As a result, the molecular composition and the structural features of "toxic Aß oligomers" have remained unresolved. Here, we have used a novel approach, hydration from gas phase coupled with isotope-edited Fourier transform infrared (FTIR) spectroscopy, to identify the prefibrillar assemblies formed by Aß and AßpE and to resolve the structures of both peptides in combination. The peptides form unusual ß-sheet oligomers stabilized by intramolecular H-bonding as opposed to intermolecular H-bonding in the fibrils. Time-dependent morphological changes in peptide assemblies have been visualized by atomic force microscopy. Aß/AßpE hetero-oligomers exert unsurpassed cytotoxic effect on PC12 cells as compared to oligomers of individual peptides or fibrils. These findings lead to a novel concept that Aß/AßpE hetero-oligomers, not just Aß or AßpE oligomers, constitute the main neurotoxic conformation. The hetero-oligomers thus present a new biomarker that may be targeted for development of more efficient diagnostic and immunotherapeutic strategies to combat AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Modelos Moleculares , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Agregación Patológica de Proteínas/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Animales , Isótopos de Carbono , Supervivencia Celular , Humanos , Enlace de Hidrógeno , Microscopía de Fuerza Atómica , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Neuronas/patología , Células PC12 , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Agregación Patológica de Proteínas/patología , Conformación Proteica en Lámina beta , Multimerización de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Terpenes make up the largest and most diverse class of natural compounds and have important commercial and medical applications. Limonene is a cyclic monoterpene (C10) present in nature as two enantiomers, (+) and (-), which are produced by different enzymes. The mechanism of production of the (-)-enantiomer has been studied in great detail, but to understand how enantiomeric selectivity is achieved in this class of enzymes, it is important to develop a thorough biochemical description of enzymes that generate (+)-limonene, as well. Here we report the first cloning and biochemical characterization of a (+)-limonene synthase from navel orange (Citrus sinensis). The enzyme obeys classical Michaelis-Menten kinetics and produces exclusively the (+)-enantiomer. We have determined the crystal structure of the apoprotein in an "open" conformation at 2.3 Å resolution. Comparison with the structure of (-)-limonene synthase (Mentha spicata), which is representative of a fully closed conformation (Protein Data Bank entry 2ONG ), reveals that the short H-α1 helix moves nearly 5 Å inward upon substrate binding, and a conserved Tyr flips to point its hydroxyl group into the active site.
Asunto(s)
Apoproteínas/química , Citrus sinensis/química , Ciclohexenos/química , Liasas Intramoleculares/química , Proteínas de Plantas/química , Proteínas Recombinantes de Fusión/química , Terpenos/química , Apoproteínas/genética , Apoproteínas/metabolismo , Dominio Catalítico , Citrus sinensis/enzimología , Clonación Molecular , Cristalografía por Rayos X , Ciclohexenos/metabolismo , Difosfatos/química , Difosfatos/metabolismo , Diterpenos/química , Diterpenos/metabolismo , Pruebas de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Liasas Intramoleculares/genética , Liasas Intramoleculares/metabolismo , Cinética , Limoneno , Mentha spicata/química , Mentha spicata/enzimología , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Dominios Proteicos , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Estereoisomerismo , Terpenos/metabolismoRESUMEN
The stereochemical course of monoterpene synthase reactions is thought to be determined early in the reaction sequence by selective binding of distinct conformations of the geranyl diphosphate (GPP) substrate. We explore here formation of early Michaelis complexes of the (+)-limonene synthase [(+)-LS] from Citrus sinensis using monofluorinated substrate analogues 2-fluoro-GPP (FGPP) and 2-fluoroneryl diphosphate (FNPP). Both are competitive inhibitors for (+)-LS with KI values of 2.4 ± 0.5 and 39.5 ± 5.2 µM, respectively. The KI values are similar to the KM for the respective nonfluorinated substrates, indicating that fluorine does not significantly perturb binding of the ligand to the enzyme. FGPP and FNPP are also substrates, but with dramatically reduced rates (kcat values of 0.00054 ± 0.00005 and 0.00024 ± 0.00002 s-1, respectively). These data are consistent with a stepwise mechanism for (+)-LS involving ionization of the allylic GPP substrate to generate a resonance-stabilized carbenium ion in the rate-limiting step. Crystals of apo-(+)-LS were soaked with FGPP and FNPP to obtain X-ray structures at 2.4 and 2.2 Å resolution, respectively. The fluorinated analogues are found anchored in the active site through extensive interactions involving the diphosphate, three metal ions, and three active-site Asp residues. Electron density for the carbon chains extends deep into a hydrophobic pocket, while the enzyme remains mostly in the open conformation observed for the apoprotein. While FNPP was found in multiple conformations, FGPP, importantly, was in a single, relatively well-defined, left-handed screw conformation, consistent with predictions for the mechanism of stereoselectivity in the monoterpene synthases.
Asunto(s)
Apoproteínas/química , Citrus sinensis/química , Ciclohexenos/química , Diterpenos/química , Inhibidores Enzimáticos/química , Liasas Intramoleculares/química , Organofosfatos/química , Proteínas de Plantas/química , Terpenos/química , Apoproteínas/antagonistas & inhibidores , Apoproteínas/genética , Apoproteínas/metabolismo , Dominio Catalítico , Citrus sinensis/enzimología , Clonación Molecular , Cristalografía por Rayos X , Ciclohexenos/metabolismo , Diterpenos/metabolismo , Pruebas de Enzimas , Inhibidores Enzimáticos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Liasas Intramoleculares/antagonistas & inhibidores , Liasas Intramoleculares/genética , Liasas Intramoleculares/metabolismo , Cinética , Ligandos , Limoneno , Modelos Moleculares , Organofosfatos/metabolismo , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fosfatos de Poliisoprenilo/química , Fosfatos de Poliisoprenilo/metabolismo , Dominios Proteicos , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Estereoisomerismo , Terpenos/metabolismoRESUMEN
Amyloid ß peptide (Aß) is causatively associated with Alzheimer's disease (AD), and N-terminally truncated and pyroglutamylated Aß peptides (AßpE) exert hypertoxic effect by an unknown mechanism. Recent evidence has identified the prefibrillar oligomers of Aß, not the fibrils, as the prevalent cytotoxic species. Structural characterization of Aß and AßpE oligomers is therefore important for better understanding of their toxic effect. Here we have used isotope-edited Fourier transform infrared (FTIR) spectroscopy to identify the conformational changes in Aß(1-42) and AßpE(3-42) upon aggregation, individually and in 1 : 1 molar combination. During the first two hours of exposure to aqueous buffer, the peptides undergo transition from mostly α-helical to mostly ß-sheet structure. Data on peptides (13)C,(15)N-labeled at K(16)L(17)V(18) or V(36)G(37)G(38)V(39) allowed construction of structural models for the monomer and early oligomers. The peptide monomer comprises a ß-hairpin that involves residues upstream of the K(16)L(17)V(18) sequence and an N-terminal α-helix. The oligomers form by non-H-bonding interactions between the ß-strands of neighboring ß-hairpins, in lateral or staggered manner, with the strands running parallel or antiparallel. Relative α-helical and ß-sheet propensities of Aß(1-42) and AßpE(3-42) depend on the ionic strength of the buffer, emphasizing the importance of ionic interactions in Aß peptide structure and aggregation. It is inferred that N-terminal modification of AßpE(3-42) affects the helix stability and thereby modulates ß-sheet oligomer formation. The data thus provide new insight into the molecular mechanism of Aß oligomerization by emphasizing the role of the N-terminal transient α-helical structure and by identifying structural constraints for molecular organization of the oligomers.
Asunto(s)
Péptidos beta-Amiloides/química , Glutamatos/química , Péptidos/química , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Dicroismo Circular , IsótoposRESUMEN
PAP248-286 is a 39-residue fragment (residues 248 to 286) derived from protease cleavage of prostatic acidic phosphatase in semen. The amyloid fibrils formed in vitro by PAP248-286 can dramatically enhance human immunodeficiency virus (HIV) infection. To our knowledge, we present the first report that the HIV-enhancing potency of fibrils formed by PAP248-286 is morphology dependent. We identified pleomorphic fibrils by transmission electron microscopy in two buffer conditions. Our solid-state NMR data showed that these fibrils consist of molecules in distinct conformations. In agreement with NMR, fluorescence measurements confirmed that they are assembled along different pathways, with distinct molecular structures. Furthermore, our cell-based infectivity tests detected distinct HIV-enhancing potencies for fibrils in distinct morphologies. In addition, our transmission electron microscopy and NMR results showed that semen-derived enhancer of viral infection fibrils formed in sodium bicarbonate buffer remain stable over time, but semen-derived enhancer of viral infection fibrils formed in phosphate buffered saline keep evolving after the initial 7 days incubation period. Given time, most of the assemblies in phosphate buffered saline will turn into elongated thin fibrils. They have similar secondary structure but different packing than thin fibrils formed initially after 7 days incubation.
Asunto(s)
Fosfatasa Ácida/farmacología , Amiloide/farmacología , VIH-1/patogenicidad , Fosfatasa Ácida/química , Amiloide/química , Línea Celular Tumoral , Humanos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Virulencia/efectos de los fármacosRESUMEN
The amyloid hypothesis causatively relates the fibrillar deposits of amyloid ß peptide (Aß) to Alzheimer's disease (AD). More recent data, however, identify the soluble oligomers as the major cytotoxic entities. Pyroglutamylated Aß (pE-Aß) is present in AD brains and exerts augmented neurotoxicity, which is believed to result from its higher ß-sheet propensity and faster fibrillization. While this concept is based on a set of experimental results, others have reported similar ß-sheet contents in unmodified and pyroglutamylated Aß, and slower aggregation of pE-Aß as compared to unmodified Aß, leaving the issue unresolved. Here, we assess the structural differences between Aß and pE-Aß peptides that may underlie their distinct cytotoxicities. Transmission electron microscopy identifies a larger number of prefibrillar aggregates of pE-Aß at early stages of aggregation and suggests that pE-Aß affects the fibrillogenesis even at low molar fractions. Circular dichroism and FTIR data indicate that while the unmodified Aß readily forms ß-sheet fibrils in aqueous media, pE-Aß displays increased α-helical and decreased ß-sheet propensity. Moreover, isotope-edited FTIR spectroscopy shows that pE-Aß reverses ß-sheet formation and hence fibrillogenesis of the unmodified Aß peptide via a prion-like mechanism. These data provide a novel structural mechanism for pE-Aß hypertoxicity; pE-Aß undergoes faster formation of prefibrillar aggregates due to its increased hydrophobicity, thus shifting the initial stages of fibrillogenesis toward smaller, hypertoxic oligomers of partial α-helical structure.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/ultraestructura , Ácido Glutámico/análogos & derivados , Priones/metabolismo , Agregación Patológica de Proteínas/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Ácido Glutámico/metabolismo , Humanos , Modelos Moleculares , Agregado de Proteínas , Agregación Patológica de Proteínas/patología , Estructura Secundaria de ProteínaRESUMEN
The cupredoxin amicyanin possesses a single tryptophan residue, Trp45. Its fluorescence is quenched when copper is bound even though it is separated by 10.1Å. Mutation of Trp45 to Ala, Phe, Leu and Lys resulted in undetectable protein expression. A W45Y amicyanin variant was isolated. The W45Y mutation did not alter the spectroscopic properties or intrinsic redox potential of amicyanin, but increased the pKa value for the pH-dependent redox potential by 0.5 units. This is due to a hydrogen-bond involving the His95 copper ligand which is present in reduced W45Y amicyanin but not in native amicyanin. The W45Y mutation significantly decreased the thermal stability of amicyanin, as determined by changes in the visible absorbance of oxidized amicyanin and in the circular dichroism spectra for oxidized, reduced and apo forms of amicyanin. Comparison of the crystal structures suggests that the decreased stability of W45Y amicyanin may be attributed to the loss of a strong interior hydrogen bond between Trp45 and Tyr90 in native amicyanin which links two of the ß-sheets that comprise the overall structure of amicyanin. Thus, Trp45 is critical for stabilizing the structure of amicyanin but it does not influence the electronic properties of the copper which quenches its fluorescence.