RESUMO
Mycoplasma genitalium, the causative agent of non-gonococcal urethritis and pelvic inflammatory disease in humans, is a small eubacterium that lacks a peptidoglycan cell wall. On the surface of its plasma membrane is the major surface adhesion complex, known as NAP that is essential for adhesion and gliding motility of the organism. Here, we have performed cryo-electron tomography of intact cells and detergent permeabilized M. genitalium cell aggregates, providing sub-tomogram averages of free and cell-attached NAPs respectively, revealing a tetrameric complex with two-fold rotational (C2) symmetry. Each NAP has two pairs of globular lobes (named α and ß lobes), arranged as a dimer of heterodimers with each lobe connected by a stalk to the cell membrane. The ß lobes are larger than the α lobes by 20%. Classification of NAPs showed that the complex can tilt with respect to the cell membrane. A protein complex containing exclusively the proteins P140 and P110, was purified from M. genitalium and was structurally characterized by negative-stain single particle EM reconstruction. The close structural similarity found between intact NAPs and the isolated P140/P110 complexes, shows that dimers of P140/P110 heterodimers are the only components of the extracellular region of intact NAPs in M. genitalium.
Assuntos
Aderência Bacteriana/fisiologia , Mycoplasma genitalium/metabolismo , Aderência Bacteriana/genética , Mycoplasma/genética , Mycoplasma/metabolismo , Infecções por Mycoplasma/microbiologia , Mycoplasma genitalium/genética , Mycoplasma genitalium/ultraestrutura , Organelas , Uretrite/microbiologiaRESUMO
The emergent human pathogen Mycoplasma genitalium, with one of the smallest genomes among cells capable of growing in axenic cultures, presents a flask-shaped morphology due to a protrusion of the cell membrane, known as the terminal organelle, that is involved in cell adhesion and motility and is an important virulence factor of this microorganism. The terminal organelle is supported by a cytoskeleton complex of about 300 nm in length that includes three substructures: the terminal button, the rod and the wheel complex. The crystal structure of the MG491 protein, a proposed component of the wheel complex, has been determined at ~3 Å resolution. MG491 subunits are composed of a 60-residue N-terminus, a central three-helix-bundle spanning about 150 residues and a C-terminal region that appears to be quite flexible and contains the region that interacts with MG200, another key protein of the terminal organelle. The MG491 molecule is a tetramer presenting a unique organization as a dimer of asymmetric pairs of subunits. The asymmetric arrangement results in two very different intersubunit interfaces between the central three-helix-bundle domains, which correlates with the formation of only ~50% of the intersubunit disulfide bridges of the single cysteine residue found in MG491 (Cys87). Moreover, M. genitalium cells with a point mutation in the MG491 gene causing the change of Cys87 to Ser present a drastic reduction in motility (as determined by microcinematography) and important alterations in morphology (as determined by electron microscopy), while preserving normal levels of the terminal organelle proteins. Other variants of MG491, designed also according to the structural information, altered significantly the motility and/or the cell morphology. Together, these results indicate that MG491 plays a key role in the functioning, organization and stabilization of the terminal organelle.
Assuntos
Adesinas Bacterianas/metabolismo , Proteínas de Bactérias/genética , Movimento Celular/fisiologia , Mycoplasma genitalium/citologia , Organelas/metabolismo , Aderência Bacteriana/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Adesão Celular , Citoesqueleto/metabolismo , Mutação/genética , Mycoplasma genitalium/genética , Mycoplasma genitalium/metabolismoRESUMO
Catalase-peroxidases (KatGs) are bifunctional enzymes exhibiting both peroxidase and substantial catalase activities. It is widely recognized from experiments that the catalatic activity of KatGs is correlated with a unique covalent adduct (M-Y-W) formed in the active site, but the exact role of this adduct was elusive up to now. Here, quantum mechanical/molecular mechanical (QM/MM) calculations and QM/MM metadynamics are employed to elucidate the molecular mechanism and the role of M-Y-W adduct in the catalase reaction. It is shown that O2 formation proceeds through a mechanism involving proton-coupled electron transfer (PCET). The M-Y-W cation radical adduct, which is close to the heme, His112 and the HOO. radical intermediate, acts as an electron sink during the PCET process. The present study also highlights the structural differences and functional similarities between KatGs and monofunctional catalases.
Assuntos
Catalase/química , Radicais Livres/química , Metionina/química , Oxigênio/química , Triptofano/química , Tirosina/química , Domínio Catalítico , Transporte de Elétrons , Heme/química , Histidina/química , Simulação de Dinâmica Molecular , Oxirredução , Peroxidases/química , Conformação Proteica , PrótonsRESUMO
Popcoen is a method for configurational entropy estimation of proteins based on machine-learning. Entropy is predicted with an artificial neural network which was trained on simulation trajectories of a large set of representative proteins. Popcoen is extremely fast compared to other approaches based on the sampling of a multitude of microstates. Consequently, Popcoen can be incorporated into a large class of protein software which currently neglects configurational entropy for performance reasons. Here, we apply Popcoen to various conformations of the Cas4 protein SSO0001 of Sulfolobus solfataricus, a protein that assembles to a decamer of known toroidal shape. We provide numerical evidence that the native state (NAT) of a SSO0001 monomer has a similar structure to the protomers of the oligomer, where NAT of the monomer is stabilized mainly entropically. Due to its large amount of configurational entropy, NAT has lower free energy than alternative conformations of very low enthalpy and solvation free-energy. Hence, SSO0001 serves as an example case where neglecting configurational entropy leads to incorrect conclusion. Our results imply that no refolding of the subunits is required during oligomerization which suggests that configurational entropy is employed by nature to largely enhance the rate of assembly.
RESUMO
The unusual Met-Tyr-Trp adduct composed of cross-linked side chains along with an associated mobile Arg is essential for catalase activity in catalase-peroxidases. In addition, acidic residues in the entrance channel, in particular an Asp and a Glu â¼7 and â¼15 Å, respectively, from the heme, significantly enhance catalase activity. The mechanism by which these channel carboxylates influence catalase activity is the focus of this work. Seventeen new variants with fewer and additional acidic residues have been constructed and characterized structurally and for enzymatic activity, revealing that their effect on activity is roughly inversely proportional to their distance from the heme and adduct, suggesting that the electrostatic potential of the heme cavity may be affected. A discrete group of protonable residues are contained within a 15 Å sphere surrounding the heme iron, and a computational analysis reveals that the pKa of the distal His112, alone, is modulated within the pH range of catalase activity by the remote acidic residues in a pattern consistent with its protonated form having a key role in the catalase reaction cycle. The electrostatic potential also impacts the catalatic reaction through its influence on the charged status of the Met-Tyr-Trp adduct.
Assuntos
Proteínas de Bactérias/metabolismo , Burkholderia pseudomallei/enzimologia , Catalase/metabolismo , Hemeproteínas/metabolismo , Histidina/química , Modelos Moleculares , Peroxidases/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Catalase/química , Catalase/genética , Domínio Catalítico , Biologia Computacional , Cristalografia por Raios X , Hemeproteínas/química , Hemeproteínas/genética , Concentração de Íons de Hidrogênio , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peroxidases/química , Peroxidases/genética , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eletricidade Estática , TitulometriaRESUMO
The mutual information expansion (MIE) represents an approximation of the configurational entropy in terms of low-dimensional integrals. It is frequently employed to compute entropies from simulation data of large systems, such as macromolecules, for which brute-force evaluation of the full configurational integral is intractable. Here, we test the validity of MIE for systems consisting of more than m = 100 degrees of freedom (dofs). The dofs are distributed according to multivariate Gaussian distributions which were generated from protein structures using a variant of the anisotropic network model. For the Gaussian distributions, we have semi-analytical access to the configurational entropy as well as to all contributions of MIE. This allows us to accurately assess the validity of MIE for different situations. We find that MIE diverges for systems containing long-range correlations which means that the error of consecutive MIE approximations grows with the truncation order n for all tractable n ⪠m. This fact implies severe limitations on the applicability of MIE, which are discussed in the article. For systems with correlations that decay exponentially with distance, MIE represents an asymptotic expansion of entropy, where the first successive MIE approximations approach the exact entropy, while MIE also diverges for larger orders. In this case, MIE serves as a useful entropy expansion when truncated up to a specific truncation order which depends on the correlation length of the system.
RESUMO
Several mycoplasmas, such as the emergent human pathogen Mycoplasma genitalium, developed a complex polar structure, known as the terminal organelle (TO), responsible for a new type of cellular motility, which is involved in a variety of cell functions: cell division, adherence to host cells, and virulence. The TO cytoskeleton is organized as a multisubunit dynamic motor, including three main ultrastructures: the terminal button, the electrodense core, and the wheel complex. Here, we describe the interaction between MG200 and MG491, two of the main components of the TO wheel complex that connects the TO with the cell body and the cell membrane. The interaction between MG200 and MG491 has a KD in the 80 nm range, as determined by surface plasmon resonance. The interface between the two partners was confined to the "enriched in aromatic and glycine residues" (EAGR) box of MG200, previously described as a protein-protein interaction domain, and to a 25-residue-long peptide from the C-terminal region of MG491 by surface plasmon resonance and NMR spectroscopy studies. An atomic description of the MG200 EAGR box binding surface was also provided by solution NMR. An M. genitalium mutant lacking the MG491 segment corresponding to the peptide reveals specific alterations in cell motility and cell morphology indicating that the MG200-MG491 interaction plays a key role in the stability and functioning of the TO.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Motores Moleculares/metabolismo , Mycoplasma genitalium/citologia , Sequência de Aminoácidos , Movimento Celular , Dicroísmo Circular , Escherichia coli/metabolismo , Teste de Complementação Genética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mutação , Organelas/metabolismo , Peptídeos/metabolismo , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de SuperfícieRESUMO
Glycogen synthase (GS) and glycogen phosphorylase (GP) are the key enzymes that control, respectively, the synthesis and degradation of glycogen, a multi-branched glucose polymer that serves as a form of energy storage in bacteria, fungi and animals. An abnormal glycogen metabolism is associated with several human diseases. Thus, GS and GP constitute adequate pharmacological targets to modulate cellular glycogen levels by means of their selective inhibition. The compound 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) is a known potent inhibitor of GP. We studied the inhibitory effect of DAB, its enantiomer LAB, and 29 DAB derivatives on the activity of rat muscle glycogen phosphorylase (RMGP) and E. coli glycogen synthase (EcGS). The isoform 4 of sucrose synthase (SuSy4) from Solanum tuberosum L. was also included in the study for comparative purposes. Although these three enzymes possess highly conserved catalytic site architectures, the DAB derivatives analysed showed extremely diverse inhibitory potential. Subtle changes in the positions of crucial residues in their active sites are sufficient to discriminate among the structural differences of the tested inhibitors. For the two Leloir-type enzymes, EcGS and SuSy4, which use sugar nucleotides as donors, the inhibitory potency of the compounds analysed was synergistically enhanced by more than three orders of magnitude in the presence of ADP and UDP, respectively. Our results are consistent with a model in which these compounds bind to the subsite in the active centre of the enzymes that is normally occupied by the glucosyl residue which is transferred between donor and acceptor substrates. The ability to selectively inhibit the catalytic activity of the key enzymes of the glycogen metabolism may represent a new approach for the treatment of disorders of the glycogen metabolism.
Assuntos
Arabinose/química , Arabinose/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glicogênio/metabolismo , Imino Furanoses/química , Imino Furanoses/farmacologia , Álcoois Açúcares/química , Álcoois Açúcares/farmacologia , Animais , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/metabolismo , Glicogênio Fosforilase/antagonistas & inibidores , Glicogênio Fosforilase/metabolismo , Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase/metabolismo , Simulação de Acoplamento Molecular , Ratos , Solanum tuberosum/efeitos dos fármacos , Solanum tuberosum/enzimologia , Solanum tuberosum/metabolismoRESUMO
Recently, it was demonstrated that bifunctional catalase-peroxidases (KatGs) are found not only in archaea and bacteria but also in lower eukaryotes. Structural studies and preliminary biochemical data of the secreted KatG from the rice pathogen Magnaporthe grisea (MagKatG2) suggested both similar and novel features when compared to those of the prokaryotic counterparts studied so far. In this work, we demonstrate the role of the autocatalytically formed redox-active Trp140-Tyr273-Met299 adduct of MagKatG2 in (i) the maintenance of the active site architecture, (ii) the catalysis of hydrogen peroxide dismutation, and (iii) the protein stability by comparing wild-type MagKatG2 with the single mutants Trp140Phe, Tyr273Phe, and Met299Ala. The impact of disruption of the covalent bonds between the adduct residues on the spectral signatures and heme cavity architecture was small. By contrast, loss of its integrity converts bifunctional MagKatG2 to a monofunctional peroxidase of significantly reduced thermal stability. It increases the accessibility of ligands due to the increased flexibility of the KatG-typical large loop 1 (LL1), which contributes to the substrate access channel and anchors at the adduct Tyr. We discuss these data with respect to those known from prokaryotic KatGs and in addition present a high-resolution structure of an oxoiron compound of MagKatG2.
Assuntos
Catalase/metabolismo , Células Eucarióticas/metabolismo , Peróxido de Hidrogênio/metabolismo , Peroxidase/metabolismo , Catalase/química , Catálise , Magnaporthe/metabolismo , Metionina/química , Metionina/metabolismo , Peroxidase/química , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato , Triptaminas/química , Triptaminas/metabolismo , Tirosina/química , Tirosina/metabolismoRESUMO
Glycogen is a polymer of α-1,4- and α-1,6-linked glucose units that provides a readily available source of energy in living organisms. Glycogen synthase (GS) and glycogen phosphorylase (GP) are the two enzymes that control, respectively, the synthesis and degradation of this polysaccharide and constitute adequate pharmacological targets to modulate cellular glycogen levels, by means of inhibition of their catalytic activity. Here we report on the synthesis and biological evaluation of a selective inhibitor that consists of an azobenzene moiety glycosidically linked to the anomeric carbon of a glucose molecule. In the ground state, the more stable (E)-isomer of the azobenzene glucoside had a slight inhibitory effect on rat muscle GP (RMGP, IC50 = 4.9 mM) and Escherichia coli GS (EcGS, IC50 = 1.6 mM). After irradiation and subsequent conversion to the (Z)-form, the inhibitory potency of the azobenzene glucoside did not significantly change for RMGP (IC50 = 2.4 mM), while its effect on EcGS increased 50-fold (IC50 = 32 µM). Sucrose synthase 4 from potatoes, a glycosyltransferase that does not operate on glycogen, was only slightly inhibited by the (E)-isomer (IC50 = 0.73 mM). These findings could be rationalized on the basis of kinetic and computer-aided docking analysis, which indicated that both isomers of the azobenzene glucoside mimic the EcGS acceptor substrate and exert their inhibitory effect by binding to the glycogen subsite in the active center of the enzyme. The ability to selectively photoregulate the catalytic activity of key enzymes of glycogen metabolism may represent a new approach for the treatment of glycogen metabolism disorders.
Assuntos
Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Glicogênio Fosforilase/metabolismo , Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase/metabolismo , Glicogênio/metabolismo , Processos Fotoquímicos , Animais , Compostos Azo/química , Compostos Azo/metabolismo , Compostos Azo/farmacologia , Sítios de Ligação , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/enzimologia , Glucosídeos/química , Glicogênio Fosforilase/química , Glicogênio Sintase/química , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Conformação Proteica , Ratos , EstereoisomerismoRESUMO
Correction for 'Selective photoregulation of the activity of glycogen synthase and glycogen phosphorylase, two key enzymes in glycogen metabolism' by Mireia Díaz-Lobo, et al., Org. Biomol. Chem., 2015, 13, 7282-7288.
RESUMO
Decarboxylation of malonyl-CoA to acetyl-CoA by malonyl-CoA decarboxylase (MCD; EC 4.1.1.9) is an essential facet in the regulation of fatty acid metabolism. The structure of human peroxisomal MCD reveals a molecular tetramer that is best described as a dimer of structural heterodimers, in which the two subunits present markedly different conformations. This molecular organization is consistent with half-of-the-sites reactivity. Each subunit has an all-helix N-terminal domain and a catalytic C-terminal domain with an acetyltransferase fold (GNAT superfamily). Intersubunit disulfide bridges, Cys-206-Cys-206 and Cys-243-Cys-243, can link the four subunits of the tetramer, imparting positive cooperativity to the catalytic process. The combination of a half-of-the-sites mechanism within each structural heterodimer and positive cooperativity in the tetramer produces a complex regulatory picture that is further complicated by the multiple intracellular locations of the enzyme. Transport into the peroxisome has been investigated by docking human MCD onto the peroxisomal import protein peroxin 5, which revealed interactions that extend beyond the C-terminal targeting motif.
Assuntos
Carboxiliases/química , Dobramento de Proteína , Motivos de Aminoácidos , Carboxiliases/genética , Carboxiliases/metabolismo , Humanos , Peroxissomos/enzimologia , Peroxissomos/genética , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico/fisiologiaRESUMO
Catalase peroxidases (KatG's) are bifunctional heme proteins that can disproportionate hydrogen peroxide (catalatic reaction) despite their structural dissimilarity with monofunctional catalases. Using X-ray crystallography and QM/MM calculations, we demonstrate that the catalatic reaction of KatG's involves deprotonation of the active-site Trp, which plays a role similar to that of the distal His in monofunctional catalases. The interaction of a nearby mobile arginine with the distal Met-Tyr-Trp essential adduct (in/out) acts as an electronic switch, triggering deprotonation of the adduct Trp.
Assuntos
Catalase/metabolismo , Peroxidases/metabolismo , Triptofano/metabolismo , Catalase/química , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática , Concentração de Íons de Hidrogênio , Modelos Moleculares , Peroxidases/química , Teoria Quântica , Triptofano/químicaRESUMO
Lipoxygenases (LOXs), which are essential in eukaryotes, have no confirmed function in prokaryotes that are devoid of polyunsaturated fatty acids. The structure of a secretable LOX from Pseudomonas aeruginosa (Pa_LOX), the first available from a prokaryote, presents significant differences with respect to eukaryotic LOXs, including a cluster of helices acting as a lid to the active center. The mobility of the lid and the structural variability of the N-terminal region of Pa_LOX was confirmed by comparing 2 crystal forms. The binding pocket contains a phosphatidylethanolamine phospholipid with branches of 18 (sn-1) and 14/16 (sn-2) carbon atoms in length. Carbon atoms from the sn-1 chain approach the catalytic iron in a manner that sheds light on how the enzymatic reaction might proceed. The findings in these studies suggest that Pa_LOX has the capacity to extract and modify unsaturated phospholipids from eukaryotic membranes, allowing this LOX to play a role in the interaction of P. aeruginosa with host cells.
Assuntos
Lipoxigenase/química , Fosfatidiletanolaminas/metabolismo , Pseudomonas aeruginosa/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Domínio Catalítico , Lipoxigenase/metabolismo , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Fosfatidiletanolaminas/química , Ligação ProteicaRESUMO
For about 30 years X-ray crystallography has been by far the most powerful approach for determining virus structures at close to atomic resolutions. Information provided by these studies has deeply and extensively enriched and shaped our vision of the virus world. In turn, the ever increasing complexity and size of the virus structures being investigated have constituted a major driving force for methodological and conceptual developments in X-ray macromolecular crystallography. Landmarks of new virus structures determinations, such as the ones from the first animal viruses or from the first membrane-containing viruses, have often been associated to methodological breakthroughs in X-ray crystallography. In this chapter we present the common ground of proteins and virus crystallography with an emphasis in the peculiarities of virus studies. For example, the solution of the phase problem, a central issue in X-ray diffraction, has benefited enormously from the presence of non-crystallographic symmetry in virus crystals.
Assuntos
Cristalografia por Raios X , Vírus/química , Animais , Humanos , Modelos MolecularesRESUMO
Transporters of the amino acid, polyamine and organocation (APC) superfamily play essential roles in cell redox balance, cancer, and aminoacidurias. The bacterial L-arginine/agmatine antiporter, AdiC, is the main APC structural paradigm and shares the "5 + 5 inverted repeat" fold found in other families like the Na(+)-coupled neurotransmitter transporters. The available AdiC crystal structures capture two states of its transport cycle: the open-to-out apo and the outward-facing Arg(+)-bound occluded. However, the role of Arg(+) during the transition between these two states remains unknown. Here, we report the crystal structure at 3.0 Å resolution of an Arg(+)-bound AdiC mutant (N101A) in the open-to-out conformation, completing the picture of the major conformational states during the transport cycle of the 5 + 5 inverted repeat fold-transporters. The N101A structure is an intermediate state between the previous known AdiC conformations. The Arg(+)-guanidinium group in the current structure presents high mobility and delocalization, hampering substrate occlusion and resulting in a low translocation rate. Further analysis supports that proper coordination of this group with residues Asn101 and Trp293 is required to transit to the occluded state, providing the first clues on the molecular mechanism of substrate-induced fit in a 5 + 5 inverted repeat fold-transporter. The pseudosymmetry found between repeats in AdiC, and in all fold-related transporters, restraints the conformational changes, in particular the transmembrane helices rearrangements, which occur during the transport cycle. In AdiC these movements take place away from the dimer interface, explaining the independent functioning of each subunit.
Assuntos
Aminoácidos/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/química , Cristalografia por Raios X , Modelos Moleculares , Conformação ProteicaRESUMO
Catalase-peroxidases or KatGs can utilize organic peroxyacids and peroxides instead of hydrogen peroxide to generate the high-valent ferryl-oxo intermediates involved in the catalase and peroxidase reactions. In the absence of peroxidatic one-electron donors, the ferryl intermediates generated with a low excess (10-fold) of peroxyacetic acid (PAA) slowly decay to the ferric resting state after several minutes, a reaction that is demonstrated in this work by both stopped-flow UV-vis absorption measurements and EPR spectroscopic characterization of Burkholderia pseudomallei KatG (BpKatG). EPR spectroscopy showed that the [Fe(IV)âO Trp330(â¢+)], [Fe(IV)âO Trp139(â¢)], and [Fe(IV)âO Trp153(â¢)] intermediates of the peroxidase-like cycle of BpKatG ( Colin, J. Wiseman, B. Switala, J. Loewen, P. C. Ivancich, A. ( 2009 ) J. Am. Chem. Soc. 131 , 8557 - 8563 ), formed with a low excess of PAA at low temperature, are also generated with a high excess (1000-fold) of PAA at room temperature. However, under high excess conditions, there is a rapid conversion to a persistent [Fe(IV)âO] intermediate. Analysis of tryptic peptides of BpKatG by mass spectrometry before and after treatment with PAA showed that specific tryptophan (including W330, W139, and W153), methionine (including Met264 of the M-Y-W adduct), and cysteine residues are either modified with one, two, or three oxygen atoms or could not be identified in the spectrum because of other undetermined modifications. It was concluded that these oxidized residues were the source of electrons used to reduce the excess of PAA to acetic acid and return the enzyme to the ferric state. Treatment of BpKatG with PAA also caused a loss of catalase activity towards certain substrates, consistent with oxidative disruption of the M-Y-W adduct, and a loss of peroxidase activity, consistent with accumulation of the [Fe(IV)âO] intermediate and the oxidative modification of the W330, W139, and W153. PAA, but not H2O2 or tert-butyl hydroperoxide, also caused subunit cross-linking.
Assuntos
Burkholderia pseudomallei/enzimologia , Catalase/química , Ácido Peracético/metabolismo , Peroxidases/química , Burkholderia pseudomallei/química , Burkholderia pseudomallei/genética , Catalase/genética , Catalase/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Modelos Moleculares , Oxirredução , Ácido Peracético/química , Peroxidases/genética , Peroxidases/metabolismoRESUMO
Most bacteria use the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the synthesis of their essential isoprenoid precursors. The absence of the MEP pathway in humans makes it a promising new target for the development of much needed new and safe antimicrobial drugs. However, bacteria show a remarkable metabolic plasticity for isoprenoid production. For example, the NADPH-dependent production of MEP from 1-deoxy-D-xylulose 5-phosphate in the first committed step of the MEP pathway is catalyzed by 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) in most bacteria, whereas an unrelated DXR-like (DRL) protein was recently found to catalyze the same reaction in some organisms, including the emerging human and animal pathogens Bartonella and Brucella. Here, we report the x-ray crystal structures of the Brucella abortus DRL enzyme in its apo form and in complex with the broad-spectrum antibiotic fosmidomycin solved to 1.5 and 1.8 Å resolution, respectively. DRL is a dimer, with each polypeptide folding into three distinct domains starting with the NADPH-binding domain, in resemblance to the structure of bacterial DXR enzymes. Other than that, DRL and DXR show a low structural relationship, with a different disposition of the domains and a topologically unrelated C-terminal domain. In particular, the active site of DRL presents a unique arrangement, suggesting that the design of drugs that would selectively inhibit DRL-harboring pathogens without affecting beneficial or innocuous bacteria harboring DXR should be feasible. As a proof of concept, we identified two strong DXR inhibitors that have virtually no effect on DRL activity.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Proteínas de Bactérias/metabolismo , Brucella abortus/enzimologia , Complexos Multienzimáticos/metabolismo , Oxirredutases/metabolismo , Terpenos/metabolismo , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Biocatálise/efeitos dos fármacos , Brucella abortus/genética , Brucella abortus/metabolismo , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Fosfomicina/análogos & derivados , Fosfomicina/química , Fosfomicina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Oxirredutases/química , Oxirredutases/genética , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Catalase-peroxidases (KatGs) are bifunctional heme enzymes widely spread in archaea, bacteria, and lower eukaryotes. Here we present the first crystal structure (1.55 Å resolution) of an eukaryotic KatG, the extracellular or secreted enzyme from the phytopathogenic fungus Magnaporthe grisea. The heme cavity of the homodimeric enzyme is similar to prokaryotic KatGs including the unique distal (+)Met-Tyr-Trp adduct (where the Trp is further modified by peroxidation) and its associated mobile arginine. The structure also revealed several conspicuous peculiarities that are fully conserved in all secreted eukaryotic KatGs. Peculiarities include the wrapping at the dimer interface of the N-terminal elongations from the two subunits and cysteine residues that cross-link the two subunits. Differential scanning calorimetry and temperature- and urea-mediated unfolding followed by UV-visible, circular dichroism, and fluorescence spectroscopy combined with site-directed mutagenesis demonstrated that secreted eukaryotic KatGs have a significantly higher conformational stability as well as a different unfolding pattern when compared with intracellular eukaryotic and prokaryotic catalase-peroxidases. We discuss these properties with respect to the structure as well as the postulated roles of this metalloenzyme in host-pathogen interactions.
Assuntos
Catalase/química , Peroxidase/química , Arginina/química , Varredura Diferencial de Calorimetria/métodos , Dicroísmo Circular , Sequência Conservada , Cristalografia por Raios X/métodos , Escherichia coli/enzimologia , Peróxido de Hidrogênio/química , Magnaporthe/enzimologia , Metaloproteínas/química , Mutagênese Sítio-Dirigida , Estresse Oxidativo , Oxigênio/química , Filogenia , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Espectrofotometria Ultravioleta/métodosRESUMO
Mycoplasma genitalium is an emerging human pathogen with the smallest genome found among self-replicating organisms. M. genitalium presents a complex cytoskeleton with a differentiated protrusion known as the terminal organelle. This polar structure plays a central role in functions essential for the virulence of the microorganism, such as motility and cell-host adhesion. A well-conserved Enriched in Aromatic and Glycine Residues motif, the EAGR box, is present in many of the proteins found in the terminal organelle. We determined the crystal structure of the globular domain from M. genitalium MG200 that contains an EAGR box. This structural information is the first at near atomic resolution for the components of the terminal organelle. The structure revealed a dimer stabilized by a compact hydrophobic core that extends throughout the dimer interface. Monomers present a new fold that contains an accurate intra-subunit symmetry relating two conspicuous hairpins. Some features, such as the domain plasticity and the presence and organization of the intra- and inter-subunit symmetry axes, support a role for the EAGR box in protein-protein interactions. Genetic, biochemical and microcinematography analyses of MG200 variants lacking the EAGR box containing domain confirm the relevant and specific association of this domain with cell motility.