RESUMEN
BpeB and BpeF are multidrug efflux transporters from Burkholderia pseudomallei that enable multidrug resistance. Here, we report the crystal structures of BpeB and BpeF at 2.94 Å and 3.0 Å resolution, respectively. BpeB was found as an asymmetric trimer, consistent with the widely-accepted functional rotation mechanism for this type of transporter. One of the monomers has a distinct structure that we interpret as an intermediate along this functional cycle. Additionally, a detergent molecule bound in a previously undescribed binding site provides insights into substrate translocation through the pathway. BpeF shares structural similarities with the crystal structure of OqxB from Klebsiella pneumoniae, where both are symmetric trimers composed of three "binding"-state monomers. The structures of BpeB and BpeF further our understanding of the functional mechanisms of transporters belonging to the HAE1-RND superfamily.
Asunto(s)
Burkholderia pseudomallei , Burkholderia pseudomallei/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Transporte Biológico , Resistencia a Múltiples Medicamentos , Sitios de Unión , Antibacterianos/farmacologíaRESUMEN
Anti-infective drug discovery is greatly facilitated by the availability of in vitro assays that are more proficient at predicting the preclinical success of screening hits. Tuberculosis (TB) drug discovery is hindered by the relatively slow growth rate of Mycobacterium tuberculosis and the use of whole-cell-based in vitro assays that are inherently time-consuming, and for these reasons, rapid, noninvasive bioluminescence-based assays have been widely used in anti-TB drug discovery and development. In this study, in vitro assays that employ autoluminescent M. tuberculosis were optimized to determine MIC, minimum bactericidal concentration (MBC), time-kill curves, activity against macrophage internalized M. tuberculosis (90% effective concentration [EC90]), and postantibiotic effect (PAE) to provide rapid and dynamic biological information. Standardization of the luminescence-based MIC, MBC, time-kill, EC90, and PAE assays was accomplished by comparing results of established TB drugs and two ClpC1-targeting TB leads, ecumicin and rufomycin, to those obtained from conventional assays and/or to previous studies. Cumulatively, the use of the various streamlined luminescence-based in vitro assays has reduced the time for comprehensive in vitro profiling (MIC, MBC, time-kill, EC90, and PAE) by 2 months. The luminescence-based in vitro MBC and EC90 assays yield time and concentration-dependent kill information that can be used for pharmacokinetic-pharmacodynamic (PK-PD) modeling. The MBC and EC90 time-kill graphs revealed a significantly more rapid bactericidal activity for ecumicin than rufomycin. The PAEs of both ecumicin and rufomycin were comparable to that of the first-line TB drug rifampin. The optimization of several nondestructive, luminescence-based TB assays facilitates the in vitro profiling of TB drug leads in an efficient manner.
Asunto(s)
Antiinfecciosos , Mycobacterium tuberculosis , Tuberculosis , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Humanos , Pruebas de Sensibilidad Microbiana , Tuberculosis/tratamiento farmacológicoRESUMEN
Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the component responsible for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has an amino acid sequence that is 36% identical to that of Streptomyces coelicolor AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomyces genus. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Å, binding properties with neutral red and deoxyadenosine were surveyed, backbone dynamics were measured, and thermal stability was assayed by circular dichroism spectroscopy. The protein is composed of four approximate repeats with a ßαßßß topology arranged radially in consecutive pairs to form two continuous eight-strand ß-sheets capped on both ends with an α-helix. The two ß-sheets intersect in the center at roughly a right angle and form two asymmetric deep "saddles" that may serve to bind ligands. Nuclear magnetic resonance chemical shift perturbation experiments show that neutral red and deoxyadenosine bind to Rv0577. Binding to deoxyadenosine is weaker with an estimated dissociation constants of 4.1 ± 0.3 mM for saddle 1. Heteronuclear steady-state {1H}-15N nuclear Overhauser effect, T1, and T2 values were generally uniform throughout the sequence with only a few modest pockets of differences. Circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C.
Asunto(s)
Proteínas Bacterianas/metabolismo , Desoxiadenosinas/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo , Rojo Neutro/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Dicroismo Circular , Cristalografía por Rayos X , Desoxiadenosinas/química , Calor/efectos adversos , Péptidos y Proteínas de Señalización Intracelular , Cinética , Ligandos , Conformación Molecular , Rojo Neutro/química , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Estabilidad Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Streptomyces coelicolor/metabolismo , Homología Estructural de ProteínaRESUMEN
AcrB is an inner membrane resistance-nodulation-cell division efflux pump and is part of the AcrAB-TolC tripartite efflux system. We have determined the crystal structure of AcrB with bound Linezolid at a resolution of 3.5 Å. The structure shows that Linezolid binds to the A385/F386 loops of the symmetric trimer of AcrB. A conformational change of a loop in the bottom of the periplasmic cleft is also observed.
Asunto(s)
Acetamidas/química , Proteínas de la Membrana Bacteriana Externa/química , Proteínas Bacterianas/química , Oxazolidinonas/química , Acetamidas/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Cristalografía por Rayos X , Farmacorresistencia Bacteriana Múltiple , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Linezolid , Modelos Moleculares , Oxazolidinonas/metabolismo , Periplasma/metabolismoRESUMEN
Ligands interacting with Mycobacterium tuberculosis recombinant proteins were identified through use of the ability of Cibacron Blue F3GA dye to interact with nucleoside/nucleotide binding proteins, and the effects of these ligands on crystallization were examined. Co-crystallization with ligands enhanced crystallization and enabled X-ray diffraction data to be collected to a resolution of atleast 2.7 Å for 5 of 10 proteins tested. Additionally, clues about individual proteins' functions were obtained from their interactions with each of a panel of ligands.
Asunto(s)
Cromatografía de Afinidad/métodos , Cristalografía por Rayos X/métodos , Nucleótidos/química , Triazinas/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Escherichia coli/química , Escherichia coli/genética , Ligandos , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , NADP Transhidrogenasas/química , NADP Transhidrogenasas/genética , Nucleótidos/genética , Mapeo de Interacción de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The ability of Mycobacterium tuberculosis (Mtb) to persist in its host may enable an evolutionary advantage for drug resistant variants to emerge. A potential strategy to prevent persistence and gain drug efficacy is to directly target the activity of enzymes that are crucial for persistence. We present a method for expedited discovery and structure-based design of lead compounds by targeting the hypoxia-associated enzyme L-alanine dehydrogenase (AlaDH). Biochemical and structural analyses of AlaDH confirmed binding of nucleoside derivatives and showed a site adjacent to the nucleoside binding pocket that can confer specificity to putative inhibitors. Using a combination of dye-ligand affinity chromatography, enzyme kinetics and protein crystallographic studies, we show the development and validation of drug prototypes. Crystal structures of AlaDH-inhibitor complexes with variations at the N6 position of the adenyl-moiety of the inhibitor provide insight into the molecular basis for the specificity of these compounds. We describe a drug-designing pipeline that aims to block Mtb to proliferate upon re-oxygenation by specifically blocking NAD accessibility to AlaDH. The collective approach to drug discovery was further evaluated through in silico analyses providing additional insight into an efficient drug development strategy that can be further assessed with the incorporation of in vivo studies.
Asunto(s)
Alanina-Deshidrogenasa , Mycobacterium tuberculosis , Alanina-Deshidrogenasa/metabolismo , Mycobacterium tuberculosis/metabolismo , Nucleósidos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Descubrimiento de DrogasRESUMEN
In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators.
Asunto(s)
Fraccionamiento Químico/métodos , Química Física/métodos , Ingeniería de Proteínas/métodos , Proteómica/métodos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The first structure for a member of the DUF3349 (PF11829) family of proteins, Rv0543c from Mycobacterium tuberculosis, has been determined using NMR-based methods and some of its biophysical properties characterized. Rv0543c is a 100 residue, 11.3 kDa protein that both size exclusion chromatography and NMR spectroscopy show to be a monomer in solution. The structure of the protein consists of a bundle of five α-helices, α1 (M1-Y16), α2 (P21-C33), α3 (S37-G52), α4 (G58-H65) and α5 (S72-G87), held together by a largely conserved group of hydrophobic amino acid side chains. Heteronuclear steady-state {¹H}-¹5N NOE, T1, and T2 values are similar through-out the sequence indicating that the backbones of the five helices are in a single motional regime. The thermal stability of Rv0543c, characterized by circular dichroism spectroscopy, indicates that Rv0543c irreversibly unfolds upon heating with an estimated melting temperature of 62.5 °C. While the biological function of Rv0543c is still unknown, the presence of DUF3349 proteins predominantly in Mycobacterium and Rhodococcus bacterial species suggests that Rv0543 may have a biological function unique to these bacteria, and consequently, may prove to be an attractive drug target to combat tuberculosis.
Asunto(s)
Proteínas Bacterianas/química , Mycobacterium tuberculosis/química , Secuencia de Aminoácidos , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Fenómenos Biofísicos , Dicroismo Circular , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Resonancia Magnética Nuclear Biomolecular , Estabilidad Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Electricidad Estática , Homología Estructural de ProteínaRESUMEN
The gene product of the open reading frame Rv3340 from Mycobacterium tuberculosis is annotated as encoding a probable O-acetylhomoserine (OAH) sulfhydrylase (MetC), an enzyme that catalyzes the last step in the biosynthesis of methionine, which is an essential amino acid in bacteria and plants. Following overexpression in Escherichia coli, the M. tuberculosis MetC enzyme was purified and crystallized using the hanging-drop vapor-diffusion method. Native diffraction data were collected from crystals belonging to space group P2(1) and were processed to a resolution of 2.1â Å.
Asunto(s)
Liasas de Carbono-Oxígeno/química , Mycobacterium tuberculosis/enzimología , Secuencia de Aminoácidos , Liasas de Carbono-Oxígeno/genética , Liasas de Carbono-Oxígeno/aislamiento & purificación , Secuencia Conservada , Cristalografía por Rayos X , Expresión Génica , Humanos , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Spinal epidural abscess (SEA) is an unusual form of spinal infection. Performing multilevel laminectomies is controversial in cases of extensive SEA considering the long surgical time and mechanical instability. Here, we report the case of an older woman with extensive SEA and poor general condition who was successfully treated with a less invasive treatment, namely skipped laminotomy using a pediatric feeding tube. A 79-year-old woman complained of progressive weakness in both legs, fever, and back pain. An extensive epidural abscess from the T3 to L5 vertebrae was observed on thoracic and lumbar magnetic resonance imaging (MRI). We performed skipped laminotomy at the T8 and T12 levels, and a 5-Fr pediatric feeding tube was advanced from the caudal level toward the rostral area and rostral level toward caudal level into the dorsal epidural space. Subsequently, regurgitation was performed with saline through the pediatric feeding tube at each level. Following this, to further irrigate the unexposed epidural abscess through laminotomy, the epidural space was washed by continuous irrigation, and the irrigation system was maintained for 48 hours. Follow-up MRI performed 3 weeks after the procedure confirmed near complete removal of the abscess in the thoracic spine, with a small residual abscess in the lumbar spine.
RESUMEN
The crystal structure of the dinB gene product from Geobacillus stearothermophilus (GsDinB) is reported at 2.5 A resolution. The dinB gene is one of the DNA-damage-induced genes and the corresponding protein, DinB, is the founding member of a Pfam family with no known function. The protein contains a four-helix up-down-down-up bundle that has previously been described in the literature in three disparate proteins: the enzyme MDMPI (mycothiol-dependent maleylpyruvate isomerase), YfiT and TTHA0303, a member of a small DUF (domain of unknown function). However, a search of the DALI structural database revealed similarities to a further 11 new unpublished structures contributed by structural genomics centers. The sequences of these proteins are quite divergent and represent several Pfam families, yet their structures are quite similar and most (but not all) seem to have the ability to coordinate a metal ion using a conserved histidine-triad motif. The structural similarities of these diverse proteins suggest that a new Pfam clan encompassing the families that share this fold should be created. The proteins that share this fold exhibit four different quaternary structures: monomeric and three different dimeric forms.
Asunto(s)
ADN Polimerasa Dirigida por ADN/química , Geobacillus stearothermophilus/enzimología , Cristalografía por Rayos X , ADN Polimerasa Dirigida por ADN/metabolismo , Modelos Moleculares , Pliegue de Proteína , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología Estructural de ProteínaRESUMEN
The crystal structure of the urease gamma subunit (UreA) from Mycobacterium tuberculosis, Rv1848, has been determined at 1.8 A resolution. The asymmetric unit contains three copies of Rv1848 arranged into a homotrimer that is similar to the UreA trimer in the structure of urease from Klebsiella aerogenes. Small-angle X-ray scattering experiments indicate that the Rv1848 protein also forms trimers in solution. The observed homotrimer and the organization of urease genes within the M. tuberculosis genome suggest that M. tuberculosis urease has the (alphabetagamma)(3) composition observed for other bacterial ureases. The gamma subunit may be of primary importance for the formation of the urease quaternary structure.
Asunto(s)
Mycobacterium tuberculosis/enzimología , Ureasa/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
We show that Cibacron Blue F3GA dye resin chromatography can be used to identify ligands that specifically interact with proteins from Mycobacterium tuberculosis, and that the identification of these ligands can facilitate structure determination by enhancing the quality of crystals. Four native Mtb proteins of the aldehyde dehydrogenase (ALDH) family were previously shown to be specifically eluted from a Cibacron Blue F3GA dye resin with nucleosides. In this study we characterized the nucleoside-binding specificity of one of these ALDH isozymes (recombinant Mtb Rv0223c) and compared these biochemical results with co-crystallization experiments with different Rv0223c-nucleoside pairings. We found that the strongly interacting ligands (NAD and NADH) aided formation of high-quality crystals, permitting solution of the first Mtb ALDH (Rv0223c) structure. Other nucleoside ligands (AMP, FAD, adenosine, GTP and NADP) exhibited weaker binding to Rv0223c, and produced co-crystals diffracting to lower resolution. Difference electron density maps based on crystals of Rv0223c with various nucleoside ligands show most share the binding site where the natural ligand NAD binds. From the high degree of similarity of sequence and structure compared to human mitochondrial ALDH-2 (BLAST Z-score = 53.5 and RMSD = 1.5 A), Rv0223c appears to belong to the ALDH-2 class. An altered oligomerization domain in the Rv0223c structure seems to keep this protein as monomer whereas native human ALDH-2 is a multimer.
Asunto(s)
Aldehído Deshidrogenasa/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Proteínas Mitocondriales/aislamiento & purificación , Mycobacterium tuberculosis/enzimología , Nucleósidos/química , Sefarosa/análogos & derivados , Aldehído Deshidrogenasa/biosíntesis , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa Mitocondrial , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Humanos , Isoenzimas/biosíntesis , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/genética , Nucleósidos/metabolismo , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Sefarosa/químicaRESUMEN
Fatty acid biosynthesis is essential for the survival of Mycobacterium tuberculosis and acetyl-coenzyme A (acetyl-CoA) is an essential precursor in this pathway. We have determined the 3-D crystal structure of M. tuberculosis citrate lyase beta-subunit (CitE), which as annotated should cleave protein bound citryl-CoA to oxaloacetate and a protein-bound CoA derivative. The CitE structure has the (beta/alpha)(8) TIM barrel fold with an additional alpha-helix, and is trimeric. We have determined the ternary complex bound with oxaloacetate and magnesium, revealing some of the conserved residues involved in catalysis. While the bacterial citrate lyase is a complex with three subunits, the M. tuberculosis genome does not contain the alpha and gamma subunits of this complex, implying that M. tuberculosis CitE acts differently from other bacterial CitE proteins. The analysis of gene clusters containing the CitE protein from 168 fully sequenced organisms has led us to identify a grouping of functionally related genes preserved in M. tuberculosis, Rattus norvegicus, Homo sapiens, and Mus musculus. We propose a novel enzymatic function for M. tuberculosis CitE in fatty acid biosynthesis that is analogous to bacterial citrate lyase but producing acetyl-CoA rather than a protein-bound CoA derivative.
Asunto(s)
Genoma Bacteriano , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Mycobacterium tuberculosis/enzimología , Oxo-Ácido-Liasas/química , Oxo-Ácido-Liasas/genética , Secuencia de Aminoácidos , Dominio Catalítico , Biología Computacional , Cristalización , Modelos Químicos , Datos de Secuencia Molecular , Complejos Multienzimáticos/fisiología , Operón , Oxo-Ácido-Liasas/fisiología , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Burkholderia cenocepacia is an opportunistic pathogen that infects cystic fibrosis patients, causing pneumonia and septicemia. B. cenocepacia has intrinsic antibiotic resistance against monobactams, aminoglycosides, chloramphenicol and fluoroquinolones that is contributed by a homologue of BpeB, which is a member of the resistance-nodulation-cell division (RND)-type multidrug-efflux transporters. Here, the cloning, overexpression, purification, construct design for crystallization and preliminary X-ray diffraction analysis of this BpeB homologue from B. cenocepacia are reported. Two truncation variants were designed to remove possible disordered regions based on comparative sequence and structural analysis to salvage the wild-type protein, which failed to crystallize. The 17-residue carboxyl-terminal truncation yielded crystals that diffracted to 3.6â Å resolution. The efflux function measured using minimal inhibitory concentration assays indicated that the truncation decreased, but did not eliminate, the efflux activity of the transporter.
Asunto(s)
Proteínas Bacterianas/química , Burkholderia cenocepacia/química , Burkholderia cenocepacia/efectos de los fármacos , Proteínas de Transporte de Membrana/química , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Farmacorresistencia Bacteriana Múltiple , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad MicrobianaRESUMEN
Structural genomics has the ambitious goal of delivering three-dimensional structural information on a genome-wide scale. Yet only a small fraction of natural proteins are suitable for structure determination because of bottlenecks such as poor expression, aggregation, and misfolding of proteins, and difficulties in solubilization and crystallization. We propose to overcome these bottlenecks by producing soluble, highly expressed proteins that are derived from and closely related to their natural homologs. Here we demonstrate the utility of this approach by using a green fluorescent protein (GFP) folding reporter assay to evolve an enzymatically active, soluble variant of a hyperthermophilic protein that is normally insoluble when expressed in Escherichia coli, and determining its structure by X-ray crystallography. Analysis of the structure provides insight into the substrate specificity of the enzyme and the improved solubility of the variant.
Asunto(s)
Genoma Bacteriano , Genómica/métodos , Nucleósido-Difosfato Quinasa/química , Ingeniería de Proteínas/métodos , Proteínas/química , Proteínas/genética , Evolución Molecular , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes , Modelos Moleculares , Nucleósido-Difosfato Quinasa/genética , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes/química , Solubilidad , Especificidad por Sustrato , Thermoproteaceae/enzimología , Thermoproteaceae/genéticaRESUMEN
Nucleoside tri-phosphates (NTP) form an important class of small molecule ligands that participate in, and are essential to a large number of biological processes. Here, we seek to identify the NTP binding proteome (NTPome) in M. tuberculosis (M.tb), a deadly pathogen. Identifying the NTPome is useful not only for gaining functional insights of the individual proteins but also for identifying useful drug targets. From an earlier study, we had structural models of M.tb at a proteome scale from which a set of 13,858 small molecule binding pockets were identified. We use a set of NTP binding sub-structural motifs derived from a previous study and scan the M.tb pocketome, and find that 1,768 proteins or 43% of the proteome can theoretically bind NTP ligands. Using an experimental proteomics approach involving dye-ligand affinity chromatography, we confirm NTP binding to 47 different proteins, of which 4 are hypothetical proteins. Our analysis also provides the precise list of binding site residues in each case, and the probable ligand binding pose. As the list includes a number of known and potential drug targets, the identification of NTP binding can directly facilitate structure-based drug design of these targets.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Genoma Bacteriano , Mycobacterium tuberculosis/metabolismo , Nucleótidos/química , Proteoma/química , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Proteínas Portadoras/clasificación , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Expresión Génica , Ontología de Genes , Ligandos , Modelos Moleculares , Anotación de Secuencia Molecular , Mycobacterium tuberculosis/genética , Nucleótidos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteoma/clasificación , Proteoma/genética , Proteoma/metabolismoRESUMEN
The three-dimensional structure of Rv2607, a putative pyridoxine 5'-phosphate oxidase (PNPOx) from Mycobacterium tuberculosis, has been determined by X-ray crystallography to 2.5 A resolution. Rv2607 has a core domain similar to known PNPOx structures with a flavin mononucleotide (FMN) cofactor. Electron density for two FMN at the dimer interface is weak despite the bright yellow color of the protein solution and crystal. The shape and size of the putative binding pocket is markedly different from that of members of the PNPOx family, which may indicate some significant changes in the FMN binding mode of this protein relative to members of the family.
Asunto(s)
Proteínas Bacterianas/química , Mycobacterium tuberculosis/enzimología , Piridoxaminafosfato Oxidasa/química , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Clonación Molecular , Secuencia Conservada , Cristalografía por Rayos X , Dimerización , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Reacción en Cadena de la Polimerasa , Pliegue de Proteína , Estructura Secundaria de Proteína , Piridoxaminafosfato Oxidasa/genética , Piridoxaminafosfato Oxidasa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
All living systems require protection against the damaging effects of reactive oxygen species. The genome of Mycobacterium tuberculosis, the cause of TB, encodes a number of peroxidases that are thought to be active against organic and inorganic peroxides, and are likely to play a key role in the ability of this organism to survive within the phagosomes of macrophages. The open reading frame Rv2238c in M.tuberculosis encodes a 153-residue protein AhpE, which is a peroxidase of the 1-Cys peroxiredoxin (Prx) family. The crystal structure of AhpE, determined at 1.87 A resolution (R(cryst)=0.179, R(free)=0.210), reveals a compact single-domain protein with a thioredoxin fold. AhpE forms both dimers and octamers; a tightly-associated dimer and a ring-like octamer, generated by crystallographic 4-fold symmetry. In this native structure, the active site Cys45 is in its oxidized, sulfenic acid (S-O-H) state. A second crystal form of AhpE, obtained after soaking in sodium bromide and refined at 1.90 A resolution (R(cryst)=0.242, R(free)=0.286), reveals the reduced structure. In this structure, a conformational change in an external loop, in two of the four molecules in the asymmetric unit, allows Arg116 to stabilise the Cys45 thiolate ion, and concomitantly closes a surface channel. This channel is identified as the likely binding site for a physiological reductant, and the conformational change is inferred to be important for the reaction cycle of AhpE.