RESUMEN
In anaerobic organisms, the decarboxylation of pyruvate, a crucial component of intermediary metabolism, is catalyzed by the metalloenzyme pyruvate: ferredoxin oxidoreductase (PFOR) resulting in the generation of low potential electrons and the subsequent acetylation of coenzyme A (CoA). PFOR is the only enzyme for which a stable acetyl thiamine diphosphate (ThDP)-based free radical reaction intermediate has been identified. The 1.87 A-resolution structure of the radical form of PFOR from Desulfovibrio africanus shows that, despite currently accepted ideas, the thiazole ring of the ThDP cofactor is markedly bent, indicating a drastic reduction of its aromaticity. In addition, the bond connecting the acetyl group to ThDP is unusually long, probably of the one-electron type already described for several cation radicals but not yet found in a biological system. Taken together, our data, along with evidence from the literature, suggest that acetyl-CoA synthesis by PFOR proceeds via a condensation mechanism involving acetyl (PFOR-based) and thiyl (CoA-based) radicals.
Asunto(s)
Coenzimas/química , Desulfovibrio/enzimología , Radicales Libres , Cetona Oxidorreductasas/química , Tiamina Pirofosfato/química , Acetilcoenzima A/metabolismo , Anaerobiosis , Sitios de Unión , Dióxido de Carbono/metabolismo , Catálisis , Fenómenos Químicos , Química Física , Coenzimas/metabolismo , Cristalización , Cristalografía por Rayos X , Dimerización , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/química , Radicales Libres/metabolismo , Cetona Oxidorreductasas/metabolismo , Conformación Molecular , Estructura Molecular , Oxidación-Reducción , Conformación Proteica , Piruvato-Sintasa , Ácido Pirúvico/metabolismo , Tiamina Pirofosfato/metabolismoRESUMEN
Ferredoxin-NADP(+) reductase (FNR) and its physiological electron donor ferredoxin (Fd) from the cyanobacterium Anabaena PCC7119 have been co-crystallized. The unit-cell parameters are a = b = 63.72, c = 158.02 A and the space group is P2(1)2(1)2(1). The crystal structure has been solved with 2.4 A resolution synchrotron data by molecular replacement, anomalous dispersion and R(min) search methods. For the computations, the crystal was treated as a merohedral twin. The asymmetric unit contains two FNR molecules and one ferredoxin molecule. The packing of the FNR molecules displays a nearly tetragonal symmetry (space group P4(3)2(1)2), whereas the ferredoxin arrangement is orthorhombic. This study provides the first crystallographic model of a dissociable complex between FNR and Fd.
Asunto(s)
Anabaena/química , Ferredoxina-NADP Reductasa/química , Ferredoxinas/química , Cristalografía por Rayos X , Modelos MolecularesRESUMEN
Ferredoxin:NADP+:reductase (FNR) catalyzes one terminal step of the conversion of light energy into chemical energy during photosynthesis. FNR uses two high energy electrons photoproduced by photosystem I (PSI) and conveyed, one by one, by a ferredoxin (Fd), to reduce NADP+ to NADPH. The reducing power of NADPH is finally involved in carbon assimilation. The interaction between oxidized FNR and Fd was studied by crystallography at 2.4 A resolution leading to a three-dimensional picture of an Fd-FNR biologically relevant complex. This complex suggests that FNR and Fd specifically interact prior to each electron transfer and disassemble upon a redox-linked conformational change of the Fd.
Asunto(s)
Anabaena , Ferredoxina-NADP Reductasa/química , Ferredoxina-NADP Reductasa/metabolismo , Ferredoxinas/química , Ferredoxinas/metabolismo , Fotosíntesis , Anabaena/química , Anabaena/enzimología , Catálisis , Cristalización , Cristalografía por Rayos X , Transporte de Electrón , Electrones , Flavina-Adenina Dinucleótido/metabolismo , Enlace de Hidrógeno , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , NADP/metabolismo , Oxidación-Reducción , Conformación Proteica , ProtonesRESUMEN
The first crystal structure of pyruvate:ferredoxin oxidoreductase to be determined has provided significant new information on its structural organization and redox chemistry. Spectroscopic analyses of a radical reaction intermediate have shed more light on its thiamin-based mechanism of catalysis. Different approaches have been used to study the interaction between the enzyme and ferredoxin, its redox partner.
Asunto(s)
Cetona Oxidorreductasas/química , Cetona Oxidorreductasas/metabolismo , Transporte de Electrón , Modelos Moleculares , Unión Proteica , Conformación Proteica , Piruvato-Sintasa , Ácido Pirúvico/metabolismo , Tiamina Pirofosfato/metabolismoRESUMEN
The structure of the homodimeric 267 kDa pyruvate:ferredoxin oxidoreductase (PFOR) of Desulfovibrio africanus was solved with data from two crystals forms, both containing two monomers per asymmetric unit. Phases were obtained from multiwavelength anomalous dispersion (MAD), solvent flattening (SF), molecular replacement (MR) using a 5 A resolution electron-density search model, multiple isomorphous replacement (MIR) and, finally, electron-density averaging (DA) procedures. It is shown how the combination of all these techniques was used to overcome problems arising from incompleteness of MAD data and weak phasing power of MIR data. A real-space refinement (RSR) procedure is described to improve MR solutions and obtain very accurate protein envelopes and non-crystallographic symmetry (NCS) transformations from 5 A resolution phase information. These were crucial for the phase extension to high resolution by DA methods.
Asunto(s)
Proteínas Bacterianas/química , Cetona Oxidorreductasas/química , Cristalización , Cristalografía por Rayos X/métodos , Desulfovibrio/enzimología , Conformación Proteica , Piruvato-Sintasa , Espectrometría por Rayos XRESUMEN
The pyruvate-ferredoxin oxidoreductase (PFOR)/ferredoxin (Fd) system of Desulfovibrio africanus has been investigated with the aim of understanding more fully protein-protein interaction and the kinetic characteristics of electron transfer between the two redox partners. D. africanus contains three Fds (Fd I, Fd II and Fd III) able to function as electron acceptors for PFOR. The complete amino acid sequence of Fd II was determined by automatic Edman degradation. It revealed a striking similarity to that of Fd I. The protein consists of 64 residues and its amino acid sequence is in agreement with a molecular mass of 6822.5 Da as measured by electrospray MS. Fd II contains five cysteine residues of which the first four (Cys11, Cys14, Cys17 and Cys54) are likely ligands for the single [4Fe-4S] cluster. A covalently cross-linked complex between PFOR and Fd I or Fd II was obtained by using a water soluble carbodiimide. This complex exhibited a stoichiometry of one ferredoxin for one PFOR subunit and is dependent on the ionic strength. The second-order rate constants for electron transfer between PFOR and Fds determined electrochemically using cyclic voltammetry are 7 x 107 M-1.s-1 for Fd I and 2 x 107 M-1.s-1 for Fd II and Fd III. The Km values of PFOR for Fd I and Fd II measured both by the electrochemical and the spectrophotometric method have been found to be 3 microM and 5 microM, respectively. The three-dimensional modelling of Fd II and surface analysis of Fd I, Fd II and PFOR suggest that a protein-protein complex is likely to be formed between aspartic acid/glutamic acid invariant residues of Fds and lysine residues surrounding the distal [4Fe-4S] cluster of PFOR. All of these studies are indicative of the involvement of electrostatic interactions between the two redox partners.
Asunto(s)
Desulfovibrio/enzimología , Ferredoxinas/química , Cetona Oxidorreductasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Carbodiimidas , Reactivos de Enlaces Cruzados , Electroquímica , Transporte de Electrón , Cinética , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Concentración Osmolar , Unión Proteica , Isoformas de Proteínas , Estructura Terciaria de Proteína , Piruvato-Sintasa , Alineación de Secuencia , Análisis de Secuencia , Electricidad EstáticaRESUMEN
For the first time, crystals of a pyruvate-ferredoxin oxidoreductase (PFOR) suitable for X-ray analysis have been obtained. This enzyme catalyzes, in anaerobic organisms, the crucial energy-yielding reaction of pyruvate decarboxylation to acetylCoA. Polyethylene glycol and divalent metal cations have been used to crystallize the PFOR from the sulfate-reducing bacterium Desulfovibrio africanus. Two different orthorhombic (P212121 ) crystal forms have been grown with unit-cell dimensions a = 86.1, b = 146.7, c = 212.5 A and a = 84.8, b = 144.9, c = 203.0 A. Both crystals diffract to 2.3 A resolution using synchrotron radiation.
Asunto(s)
Desulfovibrio/enzimología , Cetona Oxidorreductasas/química , Cetona Oxidorreductasas/aislamiento & purificación , Cristalización , Cristalografía por Rayos X , Metabolismo Energético , Cetona Oxidorreductasas/metabolismo , Piruvato-SintasaRESUMEN
Oxidative decarboxylation of pyruvate to form acetyl-coenzyme A, a crucial step in many metabolic pathways, is carried out in most aerobic organisms by the multienzyme complex pyruvate dehydrogenase. In most anaerobes, the same reaction is usually catalyzed by a single enzyme, pyruvate:ferredoxin oxidoreductase (PFOR). Thus, PFOR is a potential target for drug design against certain anaerobic pathogens. Here, we report the crystal structures of the homodimeric Desulfovibrio africanus PFOR (data to 2.3 A resolution), and of its complex with pyruvate (3.0 A resolution). The structures show that each subunit consists of seven domains, one of which affords protection against oxygen. The thiamin pyrophosphate (TPP) cofactor and the three [4Fe-4S] clusters are suitably arranged to provide a plausible electron transfer pathway. In addition, the PFOR-pyruvate complex structure shows the noncovalent fixation of the substrate before the catalytic reaction.
Asunto(s)
Cetona Oxidorreductasas/química , Ácido Pirúvico/química , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Electrones , Activación Enzimática , Cetona Oxidorreductasas/metabolismo , Modelos Moleculares , Oxígeno/química , Conformación Proteica , Piruvato-SintasaRESUMEN
The chemical sequence of the [2Fe-2S] ferredoxin from the cyanobacterium AnabaenaPCC7119 (Fd7119) and its high-resolution X-ray structures in the oxidized and reduced states have been determined. The Fd7119 sequence is identical to that of the ferredoxin from the PCC7120 strain (Fd7120). X-ray diffraction data were collected at 100 K with an oxidized trigonal Fd7119 crystal, at 1.3 A resolution, and with an orthorhombic crystal, previously reduced with dithionite and flash frozen under anaerobic conditions, at 1.17 A resolution. The two molecular models were determined by molecular replacement with the [2Fe-2S] ferredoxin from the strain PCC7120 (Rypniewski, W. R., Breiter, D. R., Benning, M. M., Wesenberg, G., Oh, B.-H., Markley, J. L., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 4126-4131.) The final R-factors are 0. 140 (for the reduced crystal) and 0.138 (for the oxidized crystal). The [2Fe-2S] cluster appears as a significantly distorted lozenge in the reduced and oxidized redox states. The major conformational difference between the two redox forms concerns the peptide bond linking Cys46 and Ser47 which points its carbonyl oxygen away from the [2Fe-2S] cluster ("CO out") in the reduced molecule and toward it ("CO in") in the oxidized one. The "CO out" conformation could be the signature of the reduction of the iron atom Fe1, which is close to the molecular surface. Superposition of the three crystallographically independent molecules shows that the putative recognition site with the physiological partner (FNR) involves charged, hydrophobic residues and invariant water molecules.
Asunto(s)
Anabaena/química , Ferredoxinas/química , Secuencia de Aminoácidos , Anabaena/genética , Ferredoxina-NADP Reductasa/química , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Conformación Proteica , Serina Endopeptidasas , Difracción de Rayos XRESUMEN
Filtration effects of turkey egg white lysozyme solution (TEWL) prior to subjecting it to crystallization conditions are investigated. Filtering TEWL solution and crystallizing it in ungelled media significantly decreased the number of conditions yielding crystals. This decrease dependent on the membrane cut-off used for filtration. From this, the postulated factors aiding in nucleation are estimated to be 0.17 microns in diameter. The existence of these factors was verified by the procedure of reversed filtration: filtered solutions passed through their inverted filter membrane a second time lead to improved crystallization results. The effect of aging of the TEWL solution prior to subjecting it to ungelled crystallization conditions was also verified. We did not find any time-dependent change in the size or the number of crystals per drop. Repeating the filtration experiments in agarose-gelled crystallization media showed that the influence of filtration on the crystallization outcome was significantly diminished. Far better crystallization results were obtained compared to ungelled media. However, there is a certain aging effect linked to filtration in gelled media. Different crystallization results were obtained depending on whether filtration was performed before or after aging and subsequent crystallization. This suggests a secondary time-dependent effect.
Asunto(s)
Filtración , Muramidasa/química , Muramidasa/aislamiento & purificación , Animales , Cristalización , Clara de Huevo , Filtración/métodos , Soluciones , Factores de Tiempo , PavosRESUMEN
The X-ray structure of the heterodimeric Ni-Fe hydrogenase from Desulfovibrio gigas, the enzyme responsible for the metabolism of molecular hydrogen, has been solved at 2.85 A resolution. The active site, which appears to contain, besides nickel, a second metal ion, is buried in the 60K subunit. The 28K subunit, which coordinates one [3Fe-4S] and two [4Fe-4S] clusters, contains an amino-terminal domain with similarities to the redox protein flavodoxin. The structure suggests plausible electron and proton transfer pathways.
Asunto(s)
Desulfovibrio/enzimología , Hidrogenasas/química , Hierro/química , Níquel/química , Secuencia de Aminoácidos , Sitios de Unión , Gráficos por Computador , Cristalografía por Rayos X , Transporte de Electrón , Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/química , Datos de Secuencia Molecular , Conformación Proteica , Procesamiento Proteico-Postraduccional , ProtonesRESUMEN
NikA, a periplasmic nickel-binding protein involved in nickel-repellent chemotaxis has been crystallized. The crystals are hexagonal with space group P6(2) (or its enantiomorph) with a = 160.3 A and c = 138.4 A and they diffract to at least 3.0 A resolution in the laboratory. NikA presents sequence homology with several periplasmic solute-binding proteins from Gram-negative bacteria.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas Bacterianas/química , Proteínas Portadoras/química , Proteínas de Escherichia coli , Escherichia coli/química , Níquel/metabolismo , Cristalización , Cristalografía por Rayos XRESUMEN
It has been previously shown that fibrinogen (FG) associates specifically with human umbilical vein and bovine aortic endothelial cells (EC) in culture and induces EC migration. In the present study, we have investigated whether the FG-EC interaction is mediated by an Arg-Gly-Asp (RGD) recognition specificity and whether EC membrane proteins related to platelet GPIIb-IIIa are involved. Highly purified radioiodinated human FG, containing no detectable fibronectin, interacted with cultured human and bovine EC in suspension in a time-dependent and specific manner. The binding was inhibited by EDTA. Two polyclonal antibodies to platelet GPIIb-IIIa, which immunoprecipitated a heterodimer molecule from EC, inhibited FG binding to EC. These same antibodies inhibited FG-induced EC migration in a dose-dependent manner as measured in a Boyden chamber. Preabsorption of the antibodies with purified platelet GPIIb-IIIa markedly reduced both inhibitory activities. A series of synthetic RGD-containing peptides inhibited FG binding to EC and FG-induced EC migration. Gly-Arg-Gly-Asp (GRGD) was the most active peptide tested in inhibiting FG binding and EC migration (ID50 of 30 microM), and conservative substitutions in the RGD sequence markedly reduced inhibitory activity (ID50 greater than 1,000 microM). These results indicate that FG binding and EC migration are events mediated by an RGD recognition specificity and that EC surface proteins immunologically related to the GPIIb-IIIa complex on platelets are involved in this recognition.