Estudio estructural y funcional del sistema de transferencia de azufre CSD ("Cysteine Sulfinate Desulfinase") de "E. coli" y proteínas con las que interacciona

La captura de azufre desde la L-Cys y su movilización son procesos esenciales para la viabilidad celular. Algunos de los componentes biológicos que necesitan de azufre son los clusters [Fe-S], los cuales se encuentran involucrados en procesos celulares cruciales. La cisteína desulfurasa es una enzima capaz de extraer los átomos de azufre del sustrato L-Cys y así donarlo a proteínas aceptoras. En la bacteria Escherichia coli se encuentran tres sistemas de movilización de azufre dependientes de la actividad cisteína desulfurasa (Isc, Suf y CSD). El sistema Isc es el sistema constitutivo, mientras que Suf es el sistema que actúa bajo condiciones de estrés oxidativo o por falta de hierro. En esta tesis doctoral, el último de los sistemas, CSD, el menos conocido de los tres, ha sido estudiado en profundidad. El sistema CSD se compone de las proteínas CsdA y CsdE, codificadas por el operón csdAE. La cisteína desulfurasa del sistema CSD, CsdA, es una enzima homodimérica dependiente de PLP, la cual actúa como un donador de azufre, mientras que CsdE es la proteína que capta los átomos de azufre de la desulfurasa, estimulando la actividad de ésta última. Estas dos proteínas forman un heterotetrámero estable. La transferencia de azufre através del sistema CSD involucra un primer paso de desulfuración de la L-cisteína, resultando en la persulfuración de la Cys358 de CsdA, y en un segundo proceso por el cual el persulfuro es cedido a la Cys61 de CsdE vía reacción de transpersulfuración. CsdE persulfurada es capaz de transferir el átomo de azufre a otras dianas. En adición, hemos estudiado proteínas claves que interaccionan con las proteínas del sistema CSD. La más destacada de estas proteínas es TcdA (anteriormente conocida como CsdL), una treonilcarbamoiladenosina deshidratasa. TcdA está codificada por un gen adyacente al operón CSD y transcrito en dirección opuesta. TcdA cataliza la ciclación de la base t6A, modificación encontrada en moléculas de ARN de transferencia (ARNt). La modificación ciclada, ct6A, predominantemente encontrada en la base A37 del stem-loop del anticodón de ARNts que reconocen codones de tipo ANN, cumple funciones de fidelidad y eficacia en el proceso de traducción..

The UlaG protein family defines novel structural and functional motifs grafted on an ancient RNase fold

Background: Bacterial populations are highly successful at colonizing new habitats and adapting to changing environmental conditions, partly due to their capacity to evolve novel virulence and metabolic pathways in response to stress conditions and to shuffle them by horizontal gene transfer (HGT). A common theme in the evolution of new functions consists of gene duplication followed by functional divergence. UlaG, a unique manganese-dependent metallo-b-lactamase (MBL) enzyme involved in L-ascorbate metabolism by commensal and symbiotic enterobacteria, provides a model for the study of the emergence of new catalytic activities from the modification of an ancient fold. Furthermore, UlaG is the founding member of the so-called UlaG-like (UlaGL) protein family, a recently established and poorly characterized family comprising divalent (and perhaps trivalent)metal-binding MBLs that catalyze transformations on phosphorylated sugars and nucleotides. Results: Here we combined protein structure-guided and sequence-only molecular phylogenetic analyses to dissect the molecular evolution of UlaG and to study its phylogenomic distribution, its relatedness with present-day UlaGL protein sequences and functional conservation. Phylogenetic analyses indicate that UlaGL sequences are present in Bacteria and Archaea, with bona fide orthologs found mainly in mammalian and plant-associated Gramnegative and Gram-positive bacteria. The incongruence between the UlaGL tree and known species trees indicates exchange by HGT and suggests that the UlaGL-encoding genes provided a growth advantage under changing conditions. Our search for more distantly related protein sequences aided by structural homology has uncovered that UlaGL sequences have a common evolutionary origin with present-day RNA processing and metabolizing MBL enzymes widespread in Bacteria, Archaea, and Eukarya. This observation suggests an ancient origin for the UlaGL family within the broader trunk of the MBL superfamily by duplication, neofunctionalization and fixation. Conclusions: Our results suggest that the forerunner of UlaG was present as an RNA metabolizing enzyme in the last common ancestor, and that the modern descendants of that ancestral gene have a wide phylogenetic distribution and functional roles. We propose that the UlaGL family evolved new metabolic roles among bacterial and possibly archeal phyla in the setting of a close association with metazoans, such as in the mammalian gastrointestinal tract or in animal and plant pathogens, as well as in environmental settings. Accordingly, the major evolutionary forces shaping the UlaGL family include vertical inheritance and lineage-specific duplication and acquisition of novel metabolic functions, followed by HGT and numerous lineage-specific gene loss events

Mechanism of sulfur transfer across protein-protein interfaces: The cysteine desulfurase model system

CsdA cysteine desulfurase (the sulfur donor) and the CsdE sulfur acceptor are involved in biological sulfur trafficking and in iron-sulfur cluster assembly in the model bacterium Escherichia coli. CsdA and CsdE form a stable complex through a polar interface that includes CsdA Cys328 and CsdE Cys61, the two residues known to be involved in the sulfur transfer reaction. Although mechanisms for the transfer of a sulfur moiety across protein-protein interfaces have been proposed based on the IscS-IscU and IscS-TusA structures, the flexibility of the catalytic cysteine loops involved has precluded a high resolution view of the active-site geometry and chemical environment for sulfur transfer. Here, we have used a combination of X-ray crystallography, solution NMR and SAXS, isothermal calorimetry, and computational chemistry methods to unravel how CsdA provides a specific recognition platform for CsdE and how their complex organizes a composite functional reaction environment. The X-ray structures of persulfurated (CsdA) and persulfurated (CsdA-CsdE) complexes reveal the crucial roles of the conserved active-site cysteine loop and additional catalytic residues in supporting the transpersulfuration reaction. A mechanistic view of sulfur transfer across protein-protein interfaces that underpins the requirement for the conserved cysteine loop to provide electrostatic stabilization for the in-transfer sulfur atom emerges from the analysis of the persulfurated (CsdA-CsdE) complex structure.BFU2008-02372/BMC, CSD 2006-23, and BFU2011-22588 to M.C., CTQ2012-36253-C03-03 and CTQ2015-66223-C2 to I.T., CTQ2015-64597-C2-1-P to J.J.B., and BFU2010-22266- C02-02 and CTQ2015-66206-C2-2-R to M.C.V. Further support for this work was obtained from the Generalitat Valenciana (ACOMP/2015/239 to I.T.) and from the European Commission FP7 ComplexINC grant (contract no. 279039) to M.C.V.Peer Reviewe

The mechanism of the transpersulfuration reaction in a cysteine desulfurase-sulfur acceptor model system

Trabajo presentado en las 1as Jornadas Españolas de Biocatálisis, celebradas en Madrid (España) del 02 al 03 de julio de 2015.Escherichia coli CsdA cysteine desulfurase (the sulfur donor) and the CsdE sulfur acceptor are involved in biological sulfur trafficking, in iron-sulfur cluster assembly, and tRNA hypermodification [1] in the model bacterium Escherichia coli. CsdA and CsdE form a stable complex through a polar interface. Although mechanisms for the transfer of a sulfur moiety across protein-protein interfaces have been proposed based on the IscS-IscU and IscS-TusA structures [2,3], the flexibility of the catalytic Cys loops involved has precluded a high resolution view of the active-site geometry and chemical environment responsible to facilitate sulfur transfer. Here, we have used a combination of X-ray crystallography, solution NMR, biophysical and computational chemistry methods to unravel how CsdA provides a specific recognition platform for CsdE and how their complex organizes a composite functional reaction environment. A mechanistic view of sulfur transfer across protein-protein interfaces emerges from the structural analysis of the CSD system

Seguimiento de la coordinación: Evaluación continua del curso 3 del Grado de Teleco de la EPS

Uno de los problemas que se ha introducido con la implantación de la evaluación continua en los nuevos grados es el exceso de carga de trabajo que conlleva la propia evaluación de las materias a lo largo del curso. Este ha sido un motivo de queja recurrente por parte de los estudiantes, los cuales se sienten estresados casi desde el comienzo del curso, llegando muchas veces a dejar de asistir durante determinados periodos a las clases para poder dedicar su atención a las evaluaciones. Esto último, a su vez, provoca quejas entre el profesorado por la no asistencia de sus estudiantes. Con el fin de mitigar esta situación, en el presente trabajo se ha realizado un estudio sobre la evaluación continua del tercer curso del Grado en Ingeniería en Sonido e Imagen en Telecomunicación de la Universidad de Alicante, y se ha consensuado un calendario de evaluación continua en el cual se intenta dosificar el esfuerzo que los estudiantes deberán realizar para sacar adelante el curso. Este trabajo está enmarcado dentro de los mecanismos de coordinación que fomenta la Escuela Politécnica Superior en sus grados

Needle in a Haystack: Targeting Specific Glucuronidases amid the Human Microbiome

11 páginasOrally administered drugs targeted against human diseases may have undesired side effects because of unforeseen interactions with enzyme activities encoded by the symbiotic microbiome in the gastrointestinal tract. A prime example is that of the common colon cancer chemotherapeutic CPT-11, a prodrug that is activated in the liver and becomes excreted as a glucuronidated end product; this inactivated product becomes reactivated in the intestine by the action of bacterial β-glucuronidases encoded by the microbiome, which remove the glucuronate moiety. Thus released, CPT-11 causes grave side effects in the intestinal epithelium leading to severe diarrhea and bloody diarrhea. A potential solution consists of a combined therapy where anticancer prodrugs as CPT-11 are supplied in conjunction with selective inhibitors against the bacterial enzymes that reactivate the liver-inactivated drug. Here we review efforts to design inhibitors against bacterial β-glucuronidases based on biochemical and structural analyses aimed at combination therapies with CPT-11 as a brilliant illustration of the complex interactions between the microbiota and current drug therapies, and discuss further examples of drugs that undergo microbiota-induced modifications that alter their pharmacological properties. Indeed, the realization that the microbiota's enzymatic repertoire has a greater than anticipated impact on therapeutic molecules given to human and animal patients has become a turning point in pharmacology and the medical sciences, and, therefore, a deeper and fuller understanding of the biotransformations of drugs catalyzed by the symbiotic flora can help the discovery of more effective treatments (and with far fewer side effects) than ever beforePeer reviewe

Insights into the inhibited form of the redox-sensitive SufE-like sulfur acceptor CsdE

17 p.-8 fig.Sulfur trafficking in living organisms relies on transpersulfuration reactions consisting in the enzyme-catalyzed transfer of S atoms via activated persulfidic S across protein-protein interfaces. The recent elucidation of the mechanistic basis for transpersulfuration in the CsdA-CsdE model system has paved the way for a better understanding of its role under oxidative stress. Herein we present the crystal structure of the oxidized, inactivated CsdE dimer at 2.4 Å resolution. The structure sheds light into the activation of the Cys61 nucleophile on its way from a solvent-secluded position in free CsdE to a fully extended conformation in the persulfurated CsdA-CsdE complex. Molecular dynamics simulations of available CsdE structures allow to delineate the sequence of conformational changes underwent by CsdE and to pinpoint the key role played by the deprotonation of the Cys61 thiol. The low-energy subunit orientation in the disulfide-bridged CsdE dimer demonstrates the likely physiologic relevance of this oxidative dead-end form of CsdE, suggesting that CsdE could act as a redox sensor in vivo.This work was supported by Spanish Instituto de Salud Carlos III (http://www.isciii.es) (PI12/01667 to MCV), Spanish Ministerio de Economía y Competitividad (http://www.mineco.gob.es/portal/site/mineco/) (PET2008_0101, BIO2009-11184, BFU2010- 22260-C02-02, and CTQ2015-66206-C2-2-R to MCV, and CTQ2015-66223-C2-2-P to IT), the Regional Government of Madrid (http://www.madrid.org/) (S2010/BD-2316 to MCV), and the European Commission (Framework Programme 7 (FP7)) (https://ec.europa.eu/research/fp7/index_en.cfm) project ComplexINC (Contract No. 279039) to MCV.Peer reviewe

The crystal structure and small-angleX-ray analysis of CsdL/TcdA reveal a new tRNA binding motif in the MoeB/E1 superfamily

22 p.-10 fig.-1 tab.-5 fig. supl.-2 tab. supl._1 text. supl.View correction information of a table and other errors at López-Estepa M, Ardá A, Savko M, Round A, Shepard WE, et al. (2015) Correction: The Crystal Structure and Small-Angle X-Ray Analysis of CsdL/TcdA Reveal a New tRNA Binding Motif in the MoeB/E1 Superfamily. PLoS ONE 10(7): e0134070. doi: 10.1371/journal.pone.0134070Cyclic N6-threonylcarbamoyladenosine (‘cyclic t6A’, ct6 A) is a non-thiolated hypermodification found in transfer RNAs (tRNAs) in bacteria, protists, fungi and plants. In bacteria and yeast cells ct6 A has been shown to enhance translation fidelity and efficiency of ANN codons by improving the faithful discrimination of aminoacylated tRNAs by the ribosome. To further the understanding of ct6A biology we have determined the high-resolution crystal structures of CsdL/TcdA in complex with AMP and ATP, an E1-like activating enzyme from Escherichia coli, which catalyzes the ATP-dependent dehydration of t6A to form ct6 A. CsdL/TcdA is a dimer whose structural integrity and dimer interface depend critically on strongly bound K+ and Na+ cations. By using biochemical assays and small-angle X-ray scattering we show that CsdL/TcdA can associate with tRNA with a 1:1 stoichiometry and with the proper position and orientation for the cyclization of t6A. Furthermore, we show by nuclear magnetic resonance that CsdL/TcdA engages in transient interactions with CsdA and CsdE, which, in the latter case, involve catalytically important residues. These short-lived interactions may underpin the precise channeling of sulfur atoms from cysteine to CsdL/TcdA as previously characterized. In summary, the combination of structural, biophysical and biochemical methods applied to CsdL/TcdA has afforded a more thorough understanding of how the structure of this E1-like enzyme has been fine tuned to accomplish ct6A synthesis on tRNAs while providing support for the notion that CsdA and CsdE are able to functionally interact with CsdL/TcdA.Ministerio de Economía y Competitividad (ES) (grants PET2008_0101, BIO2009-11184 and BFU2010-22260-C02-02 to MCV, BFU2008-02372/BMC, CONSOLIDER CSD 2006-23 and BFU2011-22588 to MC, CTQ2012-32035 to JJB), Generalitat de Catalunya (ES) (grant SGR2009-1309 to MC), the European Commission (Framework Programme 7 (FP7) projects ComplexINC No. 279039 to MCV and SILVER-GA No. 260644 to MC).Peer reviewe

Structural analysis and mutant growth properties reveal distinctive enzymatic and cellular roles for the three major L-alanine transaminases of Escherichia coli

15 p.-2 tab.-8 fig.In order to maintain proper cellular function, the metabolism of the bacterial microbiota presents several mechanisms oriented to keep a correctly balanced amino acid pool. Central components of these mechanisms are enzymes with alanine transaminase activity, pyridoxal 59-phosphate-dependent enzymes that interconvert alanine and pyruvate, thereby allowing the precise control of alanine and glutamate concentrations, two of the most abundant amino acids in the cellular amino acid pool. Here we report the 2.11-A° crystal structure of full-length AlaA from the model organism Escherichia coli, a major bacterial alanine aminotransferase, and compare its overall structure and active site composition with detailed atomic models of two other bacterial enzymes capable of catalyzing this reaction in vivo, AlaC and valine-pyruvate aminotransferase (AvtA). Apart from a narrow entry channel to the active site, a feature of this new crystal structure is the role of an active site loop that closes in upon binding of substrate-mimicking molecules, and which has only been previously reported in a plant enzyme. Comparison of the available structures indicates that beyond superficial differences, alanine aminotransferases of diverse phylogenetic origins share a universal reaction mechanism that depends on an array of highly conserved amino acid residues and is similarly regulated by various unrelated motifs. Despite this unifying mechanism and regulation, growth competition experiments demonstrate that AlaA, AlaC and AvtA are not freely exchangeable in vivo, suggesting that their functional repertoire is not completely redundant thus providing an explanation for their independent evolutionary conservation.This work was supported by the Spanish Ministry of Economy and Competitiveness (grants PET2008_0101, BIO2009-11184 and BFU2010-22260-C02-02 to MCV and BFU2008-02372/BMC, CSD 2006-23 and BFU2011-22588 to MC), the Generalitat de Catalunya (grant SGR2009-1309 to MC), the European Commission (Framework Programme 7 (FP7) projects ComplexINC No. 279039 to MCV), and the US National Institutes of Health (grant 1-R01-GM58560 to KER and AJR).Peer reviewe