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Neira J.L.,Institute Biologia Molecular y Celular | Neira J.L.,Complex Systems Physics Institute | Contreras L.M.,University of Carabobo | De Los Panos O.R.,University Miguel Hernandez | And 4 more authors.
Protein Engineering, Design and Selection | Year: 2010

Bacteriocins belong to the wide variety of antimicrobial ribosomal peptides synthesised by bacteria. Enterococci are Gram-positive, catalase-negative bacteria that produce lactic acid as the major end product of glucose fermentation. Many enterococcal strains produce bacteriocins, named enterocins. We describe in this work, the structural characterisation of the 44 residues-long enterocin EJ97, produced by Enterococcus faecalis EJ97. To this end, we have used a combined theoretical and experimental approach. First, we have characterised experimentally the conformational properties of EJ97 in solution under different conditions by using a number of spectroscopic techniques, namely fluorescence, CD, FTIR and NMR. Then, we have used several bioinformatic tools as an aid to complement the experimental information about the conformational properties of EJ97. We have shown that EJ97 is monomeric in aqueous solution and that it appears to be chiefly unfolded, save some flickering helical- or turn-like structures, probably stabilised by hydrophobic clustering. Accordingly, EJ97 does not show a cooperative sigmoidal transition when heated or upon addition of GdmCl. These conformational features are essentially pH-independent, as shown by NMR assignments at pHs 5.9 and 7.0. The computational results were puzzling, since some algorithms revealed the natively unfolded character of EJ97 (FoldIndex, the mean scaled hydropathy), whereas some others suggested the presence of ordered structure in its central region (PONDR, RONN and IUPRED). A future challenge is to produce much more experimental results to aid the development of accurate software tools for predicting disorder in proteins. © 2010 The Author. Published by Oxford University Press. All rights reserved. Source


Soriano-Maldonado P.,University of Almeria | Martinez-Gomez A.I.,University of Almeria | Andujar-Sanchez M.,University of Almeria | Neira J.L.,University Miguel Hernandez | And 5 more authors.
Applied and Environmental Microbiology | Year: 2011

Formamidases (EC 3.5.1.49) are poorly characterized proteins. In spite of this scarce knowledge, ammonia has been described as playing a central role in the pathogenesis of human pathogens such as Helicobacter pylori, for which formamidase has been shown to participate in the nitrogen metabolic pathway. Sequence analysis has revealed that at least two different groups of formamidases are classified as EC 3.5.1.49: on the one hand, the derivatives of the FmdA-AmdA superfamily, which are the best studied to date, and on the other hand, the derivatives of Helicobacter pylori AmiF. Here we present the cloning, purification, and characterization of a recombinant formamidase from Bacillus cereus CECT 5050T (BceAmiF), the second member of the AmiF subfamily to be characterized, showing new features of the enzyme further supporting its relationship with aliphatic amidases. We also present homology modeling-based mutational studies confirming the importance of the Glu140 and Tyr191 residues in the enzymatic activities of the AmiF family. Moreover, we can conclude that a second glutamate residue is critical in several members of the nitrilase superfamily, meaning that what has consistently been identified as a C-E-K triad is in fact a C-E-E-K tetrad. © 2011, American Society for Microbiology. Source


Martinez-Gomez A.I.,University of Almeria | Martinez-Gomez A.I.,Research Center en Biotecnologia Agroalimentaria | Soriano-Maldonado P.,University of Almeria | Soriano-Maldonado P.,Research Center en Biotecnologia Agroalimentaria | And 14 more authors.
Biochimie | Year: 2014

Allantoinases (allantoin amidohydrolase, E.C. 3.5.2.5) catalyze the hydrolysis of the amide bond of allantoin to form allantoic acid, in those organisms where allantoin is not the final product of uric acid degradation. Despite their importance in the purine catabolic pathway, sequences of microbial allantoinases with proven activity are scarce, and only the enzyme from Escherichia coli (AllEco) has been studied in detail in the genomic era. In this work, we report the cloning, purification and characterization of the recombinant allantoinase from Bacillus licheniformis CECT 20T (AllBali). The enzyme was a homotetramer with an apparent Tm of 62 ± 1 C. Optimal parameters for the enzyme activity were pH 7.5 and 50 C, showing apparent Km and kcat values of 17.7 ± 2.7 mM and 24.4 ± 1.5 s-1, respectively. Co2+ proved to be the most effective cofactor, inverting the enantioselectivity of AllBali when compared to that previously reported for other allantoinases. The common ability of different cyclic amidohydrolases to hydrolyze distinct substrates to the natural one also proved true for AllBali. The enzyme was able to hydrolyze hydantoin, dihydrouracil and 5-ethyl-hydantoin, although at relative rates 3-4 orders of magnitude lower than with allantoin. Mutagenesis experiments suggest that S292 is likely implicated in the binding of the allantoin ring through the carbonyl group of the polypeptide main chain, which is the common mechanism observed in other members of the amidohydrolase family. In addition, our results suggest an allosteric effect of H2O2 toward allantoinase. © 2013 Elsevier Masson SAS. All rights reserved. Source


Navarro A.,University Miguel Hernandez | Encinar J.A.,University Miguel Hernandez | Lopez-Mendez B.,CIC Biomagune | Aguado-Llera D.,University Miguel Hernandez | And 9 more authors.
Biochemistry | Year: 2012

Snapin is a 15 kDa protein present in neuronal and non-neuronal cells that has been implicated in the regulation of exocytosis and endocytosis. Protein kinase A (PKA) phosphorylates Snapin at Ser-50, modulating its function. Likewise, mutation of Cys-66, which mediates protein dimerization, impairs its cellular activity. Here, we have investigated the impact of mutating these two positions on protein oligomerization, structure, and thermal stability, along with the interaction with SNARE proteins. We found that recombinant purified Snapin in solution appears mainly as dimers in equilibrium with tetramers. The protein exhibits modest secondary structure elements and notable thermal stability. Mutation of Cys-66 to Ser abolished subunit dimerization, but not higher-order oligomers. This mutant augmented the presence of α-helical structure and slightly increased the protein thermal stability. Similarly, the S50A mutant, mimicking the unphosphorylated protein, also exhibited a higher helical secondary structure content than the wild type, along with greater thermal stability. In contrast, replacement of Ser-50 with Asp (S50D), emulating the protein-phosphorylated state, produced a loss of α-helical structure, concomitant with a decrease in protein thermal stability. In vitro, the wild type and mutants weakly interacted with SNAP-25 and the reconstituted SNARE complex, although S50D exhibited the strongest binding to the SNARE complex, consistent with the observed higher cellular activity of PKA-phosphorylated Snapin. Our observations suggest that the stronger binding of S50D to SNAREs might be due to a destabilization of tetrameric assemblies of Snapin that favor the interaction of protein dimers with the SNARE proteins. Therefore, phosphorylation of Ser-50 has an important impact on the protein structure and stability that appears to underlie its functional modulation. © 2012 American Chemical Society. Source


Romero-Beviar M.,University Miguel Hernandez | Martinez-Rodriguez S.,University of Almeria | Prieto J.,CNIO | Goormaghtigh E.,Free University of Colombia | And 7 more authors.
Protein Engineering, Design and Selection | Year: 2010

The bacterial phosphoenolpyruvate-dependent sugar phosphotransferase system is a multiprotein complex that phosphorylates and, concomitantly, transports carbohydrates across the membrane into the cell. The first protein of the cascade is a multidomain protein so-called enzyme I (EI). The N-terminal domain of EI from Streptomyces coelicolor, EINsc, responsible for the binding to the second protein in the cascade (the histidine phosphocarrier, HPr), was cloned and successfully expressed and purified. We have previously shown that EIsc binds to HPrsc with smaller affinity than other members of the EI and HPr families [Hurtado-Gómez et al. (2008) Biophys. J., 95, 1336-1348]. We think that the study of the isolated binding HPrsc domain, that is EINsc, could shed light on the small affinity value measured. Therefore, in this work we present a detailed description of the structural features of the EIN domain, as a first step towards a complete characterization of the molecular recognition process between the two proteins. We show that EINsc is a folded protein, withαhelix andβsheet structures and also random-coil conformations, as shown by circular dichroism (CD), FTIR and NMR spectroscopies. The acquisition of secondary and tertiary structures, and the burial of hydrophobic regions, occurred concomitantly at acidic pHs, but at very low pH, the domain acquired a molten-globule conformation. The EINsc protein was not very stable, with an apparent conformational free energy change upon unfolding, ΔG, of 4.1 ± 0.4 kcal mol-1, which was pH independent in the range explored (from pH 6.0 to 8.5). The thermal denaturation midpoint, which was also pH invariant, was similar to that measured in the isolated intact EIsc. Although EINsc shows thermal- and chemical denaturations that seems to follow a two-state mechanism, there is evidence of residual structure in the chemical and thermally unfolded states, as indicated by differential scanning calorimetry and CD measurements. © The Author 2010. Published by Oxford University Press. All rights reserved. Source

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