RESUMEN
FUS and TDP-43 are two self-adhesive aggregation-prone mRNA-binding proteins whose pathological mutations have been linked to neurodegeneration. While TDP-43 and FUS form reversible mRNA-rich compartments in the nucleus, pathological mutations promote their respective cytoplasmic aggregation in neurons with no apparent link between the two proteins except their intertwined function in mRNA processing. By combining analyses in cellular context and at high resolution in vitro, we unraveled that TDP-43 is specifically recruited in FUS assemblies to form TDP-43-rich subcompartments but without reciprocity. The presence of mRNA provides an additional scaffold to promote the mixing between TDP-43 and FUS. Accordingly, we also found that the pathological truncated form of TDP-43, TDP-25, which has an impaired RNA-binding ability, no longer mixes with FUS. Together, these results suggest that the binding of FUS along nascent mRNAs enables TDP-43, which is highly aggregation-prone, to mix with FUS phase to form mRNA-rich subcompartments. A functional link between FUS and TDP-43 may explain their common implication in amyotrophic lateral sclerosis.
Asunto(s)
Esclerosis Amiotrófica Lateral , Proteínas de Unión al ADN , Proteína FUS de Unión a ARN , ARN , Humanos , Esclerosis Amiotrófica Lateral/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fragmentos de Péptidos/metabolismo , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismoRESUMEN
Tuberculosis (TB) remains a global health crisis, further exacerbated by the slow pace of new treatment options, and the emergence of extreme and total drug resistance to existing drugs. The challenge to developing new antibacterial compounds with activity against Mycobacterium tuberculosis (Mtb), the causative agent of TB, is in part due to unique features of this pathogen, especially the composition and structure of its complex cell envelope. Therefore, targeting enzymes involved in cell envelope synthesis has been of major interest for anti-TB drug discovery. FAAL32 is a fatty acyl-AMP ligase involved in the biosynthesis of the cell wall mycolic acids, and a potential target for drug discovery. To rapidly advance research in this area, we initiated a drug repurposing campaign and screened a collection of 1280 approved human or veterinary drugs (Prestwick Chemical Library) using a biochemical assay that reads out FAAL32 inhibition. These efforts led to the discovery of salicylanilide closantel, and some of its derivatives as inhibitors with potent in vitro activity against M. tuberculosis. These results suggest that salicylanilide represents a potentially promising pharmacophore for the conception of novel anti-tubercular candidates targeting FAAL32 that would open new targeting opportunities. Moreover, this work illustrates the value of drug repurposing campaigns to discover new leads in challenging drug discovery fields.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Adenosina Monofosfato/uso terapéutico , Antituberculosos/química , Evaluación Preclínica de Medicamentos , Humanos , Salicilanilidas , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
SecB chaperones assist protein export in bacteria. However, certain SecB family members have diverged to become specialized toward the control of toxin-antitoxin (TA) systems known to promote bacterial adaptation to stress and persistence. In such tripartite TA-chaperone (TAC) systems, the chaperone was shown to assist folding and to prevent degradation of its cognate antitoxin, thus facilitating inhibition of the toxin. Here, we used both the export chaperone SecB of Escherichia coli and the tripartite TAC system of Mycobacterium tuberculosis as a model to investigate how generic chaperones can specialize toward the control of TA systems. Through directed evolution of SecB, we have identified and characterized mutations that specifically improve the ability of SecB to control our model TA system without affecting its function in protein export. Such a remarkable plasticity of SecB chaperone function suggests that its substrate binding surface can be readily remodeled to accommodate specific clients.
Asunto(s)
Proteínas Bacterianas/química , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Chaperonas Moleculares/química , Mycobacterium tuberculosis/genética , Sistemas Toxina-Antitoxina/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Clonación Molecular , Evolución Molecular Dirigida , Escherichia coli/metabolismo , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Cinética , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Mycobacterium tuberculosis/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The α-(1â2) branching sucrase ΔN123-GBD-CD2 is a transglucosylase belonging to glycoside hydrolase family 70 (GH70) that catalyzes the transfer ofd-glucosyl units from sucroseto dextrans or gluco-oligosaccharides via the formation of α-(1â2) glucosidic linkages. The first structures of ΔN123-GBD-CD2 in complex withd-glucose, isomaltosyl, or isomaltotriosyl residues were solved. The glucose complex revealed three glucose-binding sites in the catalytic gorge and six additional binding sites at the surface of domains B, IV, and V. Soaking with isomaltotriose or gluco-oligosaccharides led to structures in which isomaltosyl or isomaltotriosyl residues were found in glucan binding pockets located in domain V. One aromatic residue is systematically identified at the bottom of these pockets in stacking interaction with one glucosyl moiety. The carbohydrate is also maintained by a network of hydrogen bonds and van der Waals interactions. The sequence of these binding pockets is conserved and repeatedly present in domain V of several GH70 glucansucrases known to bind α-glucans. These findings provide the first structural evidence of the molecular interaction occurring between isomalto-oligosaccharides and domain V of the GH70 enzymes.
Asunto(s)
Proteínas Bacterianas/química , Oligosacáridos/química , Sacarasa/química , Proteínas Bacterianas/genética , Estructura Terciaria de Proteína , Sacarasa/genéticaRESUMEN
α-L-arabinofuranosidases are glycoside hydrolases that specifically hydrolyze non-reducing residues from arabinose-containing polysaccharides. In the case of arabinoxylans, which are the main components of hemicellulose, they are part of microbial xylanolytic systems and are necessary for complete breakdown of arabinoxylans. Glycoside hydrolase family 62 (GH62) is currently a small family of α-L-arabinofuranosidases that contains only bacterial and fungal members. Little is known about the GH62 mechanism of action, because only a few members have been biochemically characterized and no three-dimensional structure is available. Here, we present the first crystal structures of two fungal GH62 α-L-arabinofuranosidases from the basidiomycete Ustilago maydis (UmAbf62A) and ascomycete Podospora anserina (PaAbf62A). Both enzymes are able to efficiently remove the α-L-arabinosyl substituents from arabinoxylan. The overall three-dimensional structure of UmAbf62A and PaAbf62A reveals a five-bladed ß-propeller fold that confirms their predicted classification into clan GH-F together with GH43 α-L-arabinofuranosidases. Crystallographic structures of the complexes with arabinose and cellotriose reveal the important role of subsites +1 and +2 for sugar binding. Intriguingly, we observed that PaAbf62A was inhibited by cello-oligosaccharides and displayed binding affinity to cellulose although no activity was observed on a range of cellulosic substrates. Bioinformatic analyses showed that UmAbf62A and PaAbf62A belong to two distinct subfamilies within the GH62 family. The results presented here provide a framework to better investigate the structure-function relationships within the GH62 family.
Asunto(s)
Proteínas Fúngicas/química , Glicósido Hidrolasas/química , Familia de Multigenes , Podospora/enzimología , Ustilago/enzimología , Arabinosa/metabolismo , Calorimetría , Dominio Catalítico , Celulosa/metabolismo , Cristalografía por Rayos X , Proteínas Fúngicas/metabolismo , Glicósido Hidrolasas/metabolismo , Cinética , Modelos Moleculares , FilogeniaRESUMEN
The first crystal structure of Uhgb_MP, a ß-1,4-mannopyranosyl-chitobiose phosphorylase belonging to the GH130 family which is involved in N-glycan degradation by human gut bacteria, was solved at 1.85 Å resolution in the apo form and in complex with mannose and N-acetylglucosamine. SAXS and crystal structure analysis revealed a hexameric structure, a specific feature of GH130 enzymes among other glycoside phosphorylases. Mapping of the -1 and +1 subsites in the presence of phosphate confirmed the conserved Asp104 as the general acid/base catalytic residue, which is in agreement with a single-step reaction mechanism involving Man O3 assistance for proton transfer. Analysis of this structure, the first to be solved for a member of the GH130_2 subfamily, revealed Met67, Phe203 and the Gly121-Pro125 loop as the main determinants of the specificity of Uhgb_MP and its homologues towards the N-glycan core oligosaccharides and mannan, and the molecular bases of the key role played by GH130 enzymes in the catabolism of dietary fibre and host glycans.
Asunto(s)
Fosforilasas/química , Polisacáridos/química , Secuencia de Aminoácidos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Dispersión del Ángulo Pequeño , Homología de Secuencia de Aminoácido , Difracción de Rayos XRESUMEN
The universally conserved J-domain proteins (JDPs) are obligate cochaperone partners of the Hsp70 (DnaK) chaperone. They stimulate Hsp70's ATPase activity, facilitate substrate delivery, and confer specific cellular localization to Hsp70. In this work, we have identified and characterized the first functional JDP protein encoded by a bacteriophage. Specifically, we show that the ORFan gene 057w of the T4-related enterobacteriophage RB43 encodes a bona fide JDP protein, named Rki, which specifically interacts with the Escherichia coli host multifunctional DnaK chaperone. However, in sharp contrast with the three known host JDP cochaperones of DnaK encoded by E. coli, Rki does not act as a generic cochaperone in vivo or in vitro. Expression of Rki alone is highly toxic for wild-type E. coli, but toxicity is abolished in the absence of endogenous DnaK or when the conserved J-domain of Rki is mutated. Further in vivo analyses revealed that Rki is expressed early after infection by RB43 and that deletion of the rki gene significantly impairs RB43 proliferation. Furthermore, we show that mutations in the host dnaK gene efficiently suppress the growth phenotype of the RB43 rki deletion mutant, thus indicating that Rki specifically interferes with DnaK cellular function. Finally, we show that the interaction of Rki with the host DnaK chaperone rapidly results in the stabilization of the heat-shock factor σ(32), which is normally targeted for degradation by DnaK. The mechanism by which the Rki-dependent stabilization of σ(32) facilitates RB43 bacteriophage proliferation is discussed.
Asunto(s)
Bacteriófagos , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas HSP70 de Choque Térmico , Factor sigma , Proteínas Virales/genética , Proteínas Virales/metabolismo , Bacteriófagos/genética , Bacteriófagos/fisiología , Proliferación Celular , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Prueba de Complementación Genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Estructura Terciaria de Proteína/genética , Factor sigma/genética , Factor sigma/metabolismoRESUMEN
CD1e is the only human CD1 protein existing in soluble form in the late endosomes of dendritic cells, where it facilitates the processing of glycolipid antigens that are ultimately recognized by CD1b-restricted T cells. The precise function of CD1e remains undefined, thus impeding efforts to predict the participation of this protein in the presentation of other antigens. To gain insight into its function, we determined the crystal structure of recombinant CD1e expressed in human cells at 2.90-Å resolution. The structure revealed a groove less intricate than in other CD1 proteins, with a significantly wider portal characterized by a 2 Å-larger spacing between the α1 and α2 helices. No electron density corresponding to endogenous ligands was detected within the groove, despite the presence of ligands unequivocally established by native mass spectrometry in recombinant CD1e. Our structural data indicate that the water-exposed CD1e groove could ensure the establishment of loose contacts with lipids. In agreement with this possibility, lipid association and dissociation processes were found to be considerably faster with CD1e than with CD1b. Moreover, CD1e was found to mediate in vitro the transfer of lipids to CD1b and the displacement of lipids from stable CD1b-antigen complexes. Altogether, these data support that CD1e could have evolved to mediate lipid-exchange/editing processes with CD1b and point to a pathway whereby the repertoire of lipid antigens presented by human dendritic cells might be expanded.
Asunto(s)
Antígenos CD1/química , Antígenos CD1/metabolismo , Metabolismo de los Lípidos , Lípidos/química , Acilación , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Worldwide, tuberculosis is the second leading infectious killer and multidrug resistance severely hampers disease control. Mycolic acids are a unique category of lipids that are essential for viability, virulence, and persistence of the causative agent, Mycobacterium tuberculosis (Mtb). Therefore, enzymes involved in mycolic acid biosynthesis represent an important class of drug targets. We previously showed that the (3R)-hydroxyacyl-ACP dehydratase (HAD) protein HadD is dedicated mainly to the production of ketomycolic acids and plays a determinant role in Mtb biofilm formation and virulence. Here, we discovered that HAD activity requires the formation of a tight heterotetramer between HadD and HadB, a HAD unit encoded by a distinct chromosomal region. Using biochemical, structural, and cell-based analyses, we showed that HadB is the catalytic subunit, whereas HadD is involved in substrate binding. Based on HadBDMtb crystal structure and substrate-bound models, we identified determinants of the ultra-long-chain lipid substrate specificity and revealed details of structure-function relationship. HadBDMtb unique function is partly due to a wider opening and a higher flexibility of the substrate-binding crevice in HadD, as well as the drastically truncated central α-helix of HadD hotdog fold, a feature described for the first time in a HAD enzyme. Taken together, our study shows that HadBDMtb , and not HadD alone, is the biologically relevant functional unit. These results have important implications for designing innovative antivirulence molecules to fight tuberculosis, as they suggest that the target to consider is not an isolated subunit, but the whole HadBD complex.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Acido Graso Sintasa Tipo II/química , Ácidos Micólicos/metabolismo , Hidroliasas/químicaRESUMEN
ΔN(123)-glucan-binding domain-catalytic domain 2 (ΔN(123)-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1â2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of α-(1â2) linkages. The crystal structure of the apo α-(1â2) branching sucrase ΔN(123)-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-ΔN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite -1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-ΔN and ΔN(123)-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ΔN(123)-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-ΔN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ΔN(123)-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of α-(1â2) branches.
Asunto(s)
Proteínas Bacterianas/química , Leuconostoc/enzimología , Pliegue de Proteína , Sacarasa/química , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Dextranos/genética , Dextranos/metabolismo , Leuconostoc/genética , Mutación Missense , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Sacarasa/genética , Sacarasa/metabolismoRESUMEN
Amylosucrases are sucrose-utilizing α-transglucosidases that naturally catalyze the synthesis of α-glucans, linked exclusively through α1,4-linkages. Side products and in particular sucrose isomers such as turanose and trehalulose are also produced by these enzymes. Here, we report the first structural and biophysical characterization of the most thermostable amylosucrase identified so far, the amylosucrase from Deinoccocus geothermalis (DgAS). The three-dimensional structure revealed a homodimeric quaternary organization, never reported before for other amylosucrases. A sequence signature of dimerization was identified from the analysis of the dimer interface and sequence alignments. By rigidifying the DgAS structure, the quaternary organization is likely to participate in the enhanced thermal stability of the protein. Amylosucrase specificity with respect to sucrose isomer formation (turanose or trehalulose) was also investigated. We report the first structures of the amylosucrases from Deinococcus geothermalis and Neisseria polysaccharea in complex with turanose. In the amylosucrase from N. polysaccharea (NpAS), key residues were found to force the fructosyl moiety to bind in an open state with the O3' ideally positioned to explain the preferential formation of turanose by NpAS. Such residues are either not present or not similarly placed in DgAS. As a consequence, DgAS binds the furanoid tautomers of fructose through a weak network of interactions to enable turanose formation. Such topology at subsite +1 is likely favoring other possible fructose binding modes in agreement with the higher amount of trehalulose formed by DgAS. Our findings help to understand the inter-relationships between amylosucrase structure, flexibility, function, and stability and provide new insight for amylosucrase design.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Deinococcus/enzimología , Glucosiltransferasas/química , Glucosiltransferasas/metabolismo , Sacarosa/metabolismo , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Deinococcus/genética , Dimerización , Disacáridos/química , Disacáridos/metabolismo , Estabilidad de Enzimas , Fructosa/química , Fructosa/metabolismo , Glucosa/metabolismo , Glucosiltransferasas/genética , Calor , Isomerismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Especificidad por Sustrato , Sacarosa/químicaRESUMEN
Iterative saturation mutagenesis and combinatorial active site saturation focused on vicinal amino acids were used to alter the acceptor specificity of amylosucrase from Neisseria polysaccharea , a sucrose-utilizing α-transglucosidase, and sort out improved variants. From the screening of three semirational sublibraries accounting in total for 20,000 variants, we report here the isolation of three double mutants of N. polysaccharea amylosucrase displaying a spectacular specificity enhancement toward both sucrose, the donor substrate, and the allyl 2-acetamido-2-deoxy-α-D-glucopyranoside acceptor as compared to the wild-type enzyme. Such levels of activity improvement have never been reported before for this class of carbohydrate-active enzymes. X-ray structure of the best performing enzymes supported by molecular dynamics simulations showed local rigidity of the -1 subsite as well as flexibility of loops involved in active site topology, which both account for the enhanced catalytic performances of the mutants. The study well illustrates the importance of taking into account the local conformation of catalytic residues as well as protein dynamics during the catalytic process, when designing enzyme libraries.
Asunto(s)
Aminoácidos/genética , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Neisseria/enzimología , Oligosacáridos/biosíntesis , Biocatálisis , Estabilidad de Enzimas , Variación Genética/genética , Glucosiltransferasas/aislamiento & purificación , Glicosilación , Concentración de Iones de Hidrógeno , Modelos Moleculares , Mutación , Oligosacáridos/químicaRESUMEN
We report the 1.7 Å resolution crystal structure of the Lip2 lipase from Yarrowia lipolytica in its closed conformation. The Lip2 structure is highly homologous to known structures of the fungal lipase family (Thermomyces lanuginosa, Rhizopus niveus, and Rhizomucor miehei lipases). However, it also presents some unique features that are described and discussed here in detail. Structural differences, in particular in the conformation adopted by the so-called lid subdomain, suggest that the opening mechanism of Lip2 may differ from that of other fungal lipases. Because the catalytic activity of lipases is strongly dependent on structural rearrangement of this mobile subdomain, we focused on elucidating the molecular mechanism of lid motion. Using the x-ray structure of Lip2, we carried out extensive molecular-dynamics simulations in explicit solvent environments (water and water/octane interface) to characterize the major structural rearrangements that the lid undergoes under the influence of solvent or upon substrate binding. Overall, our results suggest a two-step opening mechanism that gives rise first to a semi-open conformation upon adsorption of the protein at the water/organic solvent interface, followed by a further opening of the lid upon substrate binding.
Asunto(s)
Lipasa/química , Yarrowia/enzimología , Secuencia de Aminoácidos , Arginina/metabolismo , Biocatálisis , Cristalografía por Rayos X , Disulfuros/metabolismo , Estabilidad de Enzimas , Iones , Ligandos , Datos de Secuencia Molecular , Potasio/metabolismo , Conformación Proteica , Alineación de Secuencia , Solventes , Relación Estructura-Actividad , Treonina/metabolismoRESUMEN
Some starch-degrading enzymes accommodate carbohydrates at sites situated at a certain distance from the active site. In the crystal structure of barley alpha-amylase 1, oligosaccharide is thus bound to the 'sugar tongs' site. This site on the non-catalytic domain C in the C-terminal part of the molecule contains a key residue, Tyr380, which has numerous contacts with the oligosaccharide. The mutant enzymes Y380A and Y380M failed to bind to beta-cyclodextrin-Sepharose, a starch-mimic resin used for alpha-amylase affinity purification. The K(d) for beta-cyclodextrin binding to Y380A and Y380M was 1.4 mm compared to 0.20-0.25 mm for the wild-type, S378P and S378T enzymes. The substitution in the S378P enzyme mimics Pro376 in the barley alpha-amylase 2 isozyme, which in spite of its conserved Tyr378 did not bind oligosaccharide at the 'sugar tongs' in the structure. Crystal structures of both wild-type and S378P enzymes, but not the Y380A enzyme, showed binding of the pseudotetrasaccharide acarbose at the 'sugar tongs' site. The 'sugar tongs' site also contributed importantly to the adsorption to starch granules, as Kd = 0.47 mg.mL(-1) for the wild-type enzyme increased to 5.9 mg.mL(-1) for Y380A, which moreover catalyzed the release of soluble oligosaccharides from starch granules with only 10% of the wild-type activity. beta-cyclodextrin both inhibited binding to and suppressed activity on starch granules for wild-type and S378P enzymes, but did not affect these properties of Y380A, reflecting the functional role of Tyr380. In addition, the Y380A enzyme hydrolyzed amylose with reduced multiple attack, emphasizing that the 'sugar tongs' participates in multivalent binding of polysaccharide substrates.
Asunto(s)
Hordeum/enzimología , alfa-Amilasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Dominio Catalítico , Cristalografía por Rayos X , Cartilla de ADN , Hidrólisis , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Resonancia por Plasmón de Superficie , alfa-Amilasas/química , alfa-Amilasas/genética , beta-Ciclodextrinas/metabolismoRESUMEN
Amylosucrase from Neisseria polysaccharea naturally catalyzes the synthesis of α-1,4 glucans from sucrose. The product profile is quite polydisperse, ranging from soluble chains called maltooligosaccharides to high-molecular weight insoluble amylose. This enzyme was recently subjected to engineering of its active site to enable recognition of non-natural acceptor substrates. Libraries of variants were constructed and screened on sucrose, allowing the identification of a mutant that showed a 6-fold enhanced activity toward sucrose compared to the wild-type enzyme. Furthermore, its product profile was unprecedented, as only soluble maltooligosaccharides of controlled size chains (2Asunto(s)
Glucosiltransferasas/genética
, Neisseria/enzimología
, Oligosacáridos/biosíntesis
, Ingeniería de Proteínas
, Glucosiltransferasas/metabolismo
, Sacarosa
RESUMEN
TorD is the cytoplasmic chaperone involved in the maturation of the molybdoenzyme TorA prior to the translocation of the folded protein into the periplasm. The X-ray structure at 2.4 A resolution of the TorD dimer reveals extreme domain swapping between the two subunits. The all-helical architecture of the globular domains within the intertwined molecular dimer shows no similarity with known protein structures. According to sequence similarities, this new fold probably represents the architecture of the chaperones associated with the bacterial DMSO/TMAO reductases and also that of proteins of yet unknown functions. The occurrence of multiple oligomeric forms and the chaperone activity of both monomeric and dimeric TorD raise questions about the possible biological role of domain swapping in this protein.
Asunto(s)
Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Shewanella/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Familia de Multigenes , Pliegue de Proteína , Estructura Terciaria de Proteína , Shewanella/metabolismoRESUMEN
Several bacteria use trimethylamine N-oxyde (TMAO) as an exogenous electron acceptor for anaerobic respiration. This metabolic pathway involves expression of the tor operon that codes for a periplasmic molybdopterin-containing reductase of the DMSO/TMAO family, a pentahemic c-type cytochrome, and the TorD cytoplasmic chaperone, possibly required for acquisition of the molybdenum cofactor and translocation of the reductase by the twin-arginine translocation system. In this report, we show that the TorD chaperone from Shewanella massilia forms multiple and stable oligomeric species. The monomeric, dimeric, and trimeric forms were purified to homogeneity and characterized by analytical ultracentrifugation. Small-angle X-ray scattering (SAXS) and preliminary diffraction data indicated that the TorD dimer is made of identical protein modules of similar size to the monomeric species. Interconversion of the native oligomeric forms occurred at acidic pH value. In this condition, ANS fluorescence indicates a non-native conformation of the polypeptide chain in which, according to the circular dichroism spectra, the alpha-helical content is similar to that of the native species. Surface plasmon resonance showed that both the monomeric and dimeric species bind the mature TorA enzyme, but that the dimer binds its target protein more efficiently. The possible biologic significance of these oligomers is discussed in relation to the chaperone activity of TorD, and to the ability of another member of the TorD family to bind the Twin Arginine leader sequences of the precursor of DMSO/TMAO reductases.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Escherichia coli , Chaperonas Moleculares/química , Oxidorreductasas N-Desmetilantes/metabolismo , Shewanella/química , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dicroismo Circular , Cristalografía por Rayos X , Dimerización , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Cuaternaria de Proteína , Shewanella/metabolismo , Espectrometría de Fluorescencia , Resonancia por Plasmón de SuperficieRESUMEN
The glucose isomerase gene (xylA) from the Streptomyces sp. SK strain encodes a 386-amino-acid protein (42.7 kDa) showing extensive identities with many other bacterial glucose isomerases. We have shown by gel filtration chromatography and SDS-PAGE analysis that the purified recombinant glucose isomerase (SKGI) is a 180 kDa tetramer of four 43 kDa subunits. Sequence inspection revealed that this protein, present some special characteristics like the abundance of hydrophobic residues and some original amino-acid substitutions, which distinguish SKGI from the other GIs previously reported. The presence of an Ala residue at position 103 in SKGI is especially remarkable, since the same amino-acid was found at the equivalent position in the extremely thermostable GIs from Thermus thermophilus and Thermotoga neapolitana; whereas a Gly was found in the majority of less thermostable GIs from Streptomyces. The Ala103Gly mutation, introduced in SKGI, significantly decreases the half-life time at 90 degrees C from 80 to 50 min and also shifts the optimum pH from 6.5 to 7.5. This confirms the implication of the Ala103 residue on SKGI thermostability and activity at low pH. A homology model of SKGI based on the SOGI (that of Streptomyces olivochromogenes) crystal structure has been constructed in order to understand the mutational effects on a molecular scale. Hence, the Ala103Gly mutation, affecting enzyme properties, is presumed to increase molecular flexibility and to destabilize, in particular at elevated temperature, the 91-109 loop that includes the important catalytic residue, Phe94.
Asunto(s)
Alanina/metabolismo , Isomerasas Aldosa-Cetosa/química , Isomerasas Aldosa-Cetosa/aislamiento & purificación , Streptomyces/enzimología , Ácidos/farmacología , Alanina/química , Alanina/genética , Isomerasas Aldosa-Cetosa/genética , Isomerasas Aldosa-Cetosa/metabolismo , Secuencia de Aminoácidos , Estabilidad de Enzimas/efectos de los fármacos , Calor , Concentración de Iones de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Análisis de Secuencia , Streptomyces/genéticaRESUMEN
The yeast Saccharomyces cerevisiae IMA multigene family encodes four isomaltases sharing high sequence identity from 65% to 99%. Here, we explore their functional diversity, with exhaustive in-vitro characterization of their enzymological and biochemical properties. The four isoenzymes exhibited a preference for the α-(1,6) disaccharides isomaltose and palatinose, with Michaëlis-Menten kinetics and inhibition at high substrates concentration. They were also able to hydrolyze trisaccharides bearing an α-(1,6) linkage, but also α-(1,2), α-(1,3) and α-(1,5) disaccharides including sucrose, highlighting their substrate ambiguity. While Ima1p and Ima2p presented almost identical characteristics, our results nevertheless showed many singularities within this protein family. In particular, Ima3p presented lower activities and thermostability than Ima2p despite only three different amino acids between the sequences of these two isoforms. The Ima3p_R279Q variant recovered activity levels of Ima2p, while the Leu-to-Pro substitution at position 240 significantly increased the stability of Ima3p and supported the role of prolines in thermostability. The most distant protein, Ima5p, presented the lowest optimal temperature and was also extremely sensitive to temperature. Isomaltose hydrolysis by Ima5p challenged previous conclusions about the requirement of specific amino acids for determining the specificity for α-(1,6) substrates. We finally found a mixed inhibition by maltose for Ima5p while, contrary to a previous work, Ima1p inhibition by maltose was competitive at very low isomaltose concentrations and uncompetitive as the substrate concentration increased. Altogether, this work illustrates that a gene family encoding proteins with strong sequence similarities can lead to enzyme with notable differences in biochemical and enzymological properties.
RESUMEN
The staphylococcal bipartite leukotoxins and the homoheptameric alpha-toxin belong to the same family of beta-barrel pore-forming toxins despite slight differences. In the alpha-toxin pore, the N-terminal extremity of each protomer interacts as a deployed latch with two consecutive protomers in the vicinity of the pore lumen. N-terminal extremities of leukotoxins as seen in their three-dimensional structures are heterogeneous in length and take part in the beta-sandwich core of soluble monomers. Hence, the interaction of these N-terminal extremities within structures of adjacent monomers is questionable. We show here that modifications of their N-termini by two different processes, using fusion with glutathione S-transferase (GST) and bridging of the N-terminal extremity to the adjacent beta-sheet via disulphide bridges, are not deleterious for biological activity. Therefore, bipartite leukotoxins do not need a large extension of their N-terminal extremities to form functional pores, thus illustrating a microheterogeneity of the structural organizations between bipartite leukotoxins and alpha-toxin.