{"id":117779,"date":"2015-03-07T00:00:00","date_gmt":"2015-03-07T00:00:00","guid":{"rendered":""},"modified":"2015-03-07T00:00:00","modified_gmt":"2015-03-07T00:00:00","slug":"estudio-de-la-lisina-epsilon-oxidasa-loda-sintetizada-por-marinomonas-mediterranea-papel-de-la-flavoprotea%c2%adna-lodb-en-la-generacion-del-cofactor-quinonico-de-loda-mediante-modificacion-post-tra","status":"publish","type":"post","link":"https:\/\/www.deberes.net\/tesis\/bioquimica\/estudio-de-la-lisina-epsilon-oxidasa-loda-sintetizada-por-marinomonas-mediterranea-papel-de-la-flavoprotea%c2%adna-lodb-en-la-generacion-del-cofactor-quinonico-de-loda-mediante-modificacion-post-tra\/","title":{"rendered":"Estudio de la lisina-\u00e9psilon-oxidasa loda sintetizada por marinomonas mediterr\u00e1nea. papel de la flavoprote\u00edna lodb en la generaci\u00f3n del cofactor quin\u00f3nico de loda, mediante modificaci\u00f3n post-traduccional."},"content":{"rendered":"<h2>Tesis doctoral de <strong> Mar\u00eda  Dolores Chacon Verdu <\/strong><\/h2>\n<p>Las l-amino\u00e1cido oxidasas (laos) son enzimas que oxidan amino\u00e1cidos generando el ceto\u00e1cido correspondiente, amonio y per\u00f3xido de hidr\u00f3geno. Generalmente, estas enzimas tienen una flavina como cofactor. En la bacteria marina marinomonas mediterranea se han descrito las dos primeras laos que poseen un cofactor quin\u00f3nico. Este tipo de cofactores se generan mediante modificaci\u00f3n post-traduccional de amino\u00e1cidos. La primera lao descrita en m. Mediterranea, loda, es una novedosa l-lisina-\u00e9psilon-oxidasa (ec 1.4.3.20). De forma paralela a la realizaci\u00f3n de este trabajo, se ha descrito goxa, una glicina oxidasa con caracter\u00edsticas distintas a las glicinas oxidasas descritas previamente en otros organismos. Ambas prote\u00ednas son codificadas en operones. En el caso del oper\u00f3n lod, inmediatamente despu\u00e9s al gen loda, se encuentra el gen lodb que codifica la flavoprote\u00edna lodb. Datos previos indicaban que en m. Mediterranea ambas prote\u00ednas son necesarias para obtener loda activa. El objetivo de este trabajo ha sido estudiar el papel de lodb en la generaci\u00f3n del cofactor quin\u00f3nico de loda. As\u00ed mismo, se han abordado por primera vez estudios de relaci\u00f3n estructura\/funci\u00f3n de loda para determinar residuos implicados en el proceso de generaci\u00f3n del cofactor quin\u00f3nico y en la actividad enzim\u00e1tica.   el estudio presentado en esta memoria se ha llevado a cabo principalmente mediante expresi\u00f3n recombinante, solas o en combinaci\u00f3n, en escherichia coli de las distintas prote\u00ednas de los operones que codifican las laos. Las prote\u00ednas recombinantes se analizaron mediante espectrometr\u00eda de masas para detectar la masa molecular de la prote\u00edna completa y de los p\u00e9ptidos generados por digesti\u00f3n enzim\u00e1tica, con el fin de identificar los residuos que son modificados post-traduccionalmente durante la generaci\u00f3n del cofactor quin\u00f3nico. Adicionalmente, el papel de los residuos seleccionados por estar conservados en loda y prote\u00ednas similares o por su localizaci\u00f3n estructural, se ha estudiado mediante mediante mutag\u00e9nesis dirigida. Finalmente, se ha realizado un an\u00e1lisis comparativo de las prote\u00ednas del oper\u00f3n lod y gox.   se ha determinado que loda recombinante posee como cofactor ciste\u00edna triptofilquinona (ctq) generado por modificaci\u00f3n de los residuos cys516 y trp581. La expresi\u00f3n de loda en ausencia de lodb, genera una forma precursora de loda, a la que se ha denominado preloda, que no muestra actividad enzim\u00e1tica y est\u00e1 monohidroxilada en trp581. Este resultado indica que la s\u00edntesis de la prote\u00edna activa requiere la flavoprote\u00edna lodb que actuar\u00eda sobre preloda. Por su parte, los experimentos de mutag\u00e9nesis han permitido determinar que el residuo conservado asp512 es esencial en la generaci\u00f3n de preloda. Otros residuos analizados importantes para la actividad est\u00e1n localizados en la entrada del canal del centro activo de loda o en la proximidad del cofactor. Las deleciones realizadas en el extremo c-terminal sugieren que esa zona podr\u00eda participar en la generaci\u00f3n de la forma activa de loda.   mediante an\u00e1lisis bioinform\u00e1tico se ha detectado en genomas microbianos que los operones que codifican prote\u00ednas similares a loda generalmente contienen, adem\u00e1s del gen similar a loda, un segundo gen que codifica una hipot\u00e9tica flavoprote\u00edna similar a lodb. El estudio del oper\u00f3n gox ha revelado que, al igual que ocurre con loda, cuando se expresa goxa en ausencia de goxb, se detecta una forma intermedia monohidroxilada sin actividad enzim\u00e1tica que se ha denominado pregoxa, por su similitud con preloda. Por \u00faltimo, se ha demostrado que las flavoprote\u00ednas que acompa\u00f1an a loda y goxa no son intercambiables, indicando una alta especificidad de la quinooxidasa que modifica.   este trabajo supone un avance en el estudio de la nueva familia de laos con cofactor quin\u00f3nico. En concreto, se ha puesto de manifiesto un mecanismo novedoso de modificaci\u00f3n post-traduccional de prote\u00ednas, ya que con anterioridad no se hab\u00eda descrito la participaci\u00f3n de flavoprote\u00ednas en este tipo de procesos.    l-amino acid oxidases (laos) oxidize amino acids releasing ammonium, hydrogen peroxide and the corresponding alpha keto acid. The marine bacterium marinomonas mediterranea synthesizes two novel laos which contain a quinone cofactor, in contrast to other laos that contain a flavin cofactor. Quinone cofactors are generated by post-translational modification of amino acid residues. The first lao described in m. Mediterranea, loda, is a novel l-lysine-epsilon-oxidase (ec 1.4.3.20). During the realization of this work, it has been described goxa, a glycine oxidase which is different from the glycine oxidases previously described in other organisms. Both proteins are encoded in operons containing two genes. The operon lod contains immediately downstream to the gene coding for loda, a second gene coding for the flavoprotein lodb. Previous data have demonstrated that both proteins are required for the generation of the active form of loda in m. Mediterranea. The aim of this work has been to study the role of lodb in the generation of the quinone cofactor of loda. In addition, studies about structure\/function relationship of loda has been carried out for the first time with the aim of determining the residues involved in the biogenesis of the quinone cofactor and the enzymatic activity of loda.  this work has been carried out by heterologous expression in escherichia coli of the different genes coding for either loda or goxa, alone or in combination with the genes encoding lodb or goxb. In order to identify which residues are post-translationally modified, the recombinant proteins were analyzed by mass spectrometry to determine the molecular mass of the complete protein and that of the peptides generated by enzymatic digestion. In addition, the role of residues conserved in loda and loda-like proteins has been studied by site directed mutagenesis. Finally, a comparative analysis of protein encoded by lod and gox operons has been also performed.  it has been determined that recombinant loda contains a cysteine tryptophyl quinone cofactor (ctq) which is generated by post-translational modification of residues cys516 and trp581. When loda is expressed in absence of lodb, an inactive monohydroxylated intermediate of loda, named preloda is generated. It is. This result indicates that lodb is required to obtain active loda and that preloda would be its substrate. A critical role in the generation of this precursor of a conserved asp residue has been revealed by site directed mutagenesis. Other important residues to obtain enzymatic activity are located at the entrance of the active site of loda or in close vicinity to the quinone cofactor. Moreover, deletions in c-terminal region of loda suggest a role of that region in the generation of the active form of loda.  using bioinformatic analysis it has detected that operons encoding loda-like proteins in microbial genomes usually contain a second gene encoding a hypothetical lodb-like flavoprotein. The analysis of the recombinant synthesis of goxa in absence of goxb has revealed, similarly to loda, a soluble inactive precursor with partial post-translational modifications. This precursor has been named pregoxa because of its similarity to preloda. Finally, it has been demonstrated that the roles of lodb and goxb are not interchangeable and each flavoprotein acts specifically on its partner protein.   this study gets insights in a novel family of laos with quinone cofactors. The data presented in this work have revealed a new mechanism of post-translational modification of proteins involving a flavoprotein.<\/p>\n<p>&nbsp;<\/p>\n<h3>Datos acad\u00e9micos de la tesis doctoral \u00ab<strong>Estudio de la lisina-\u00e9psilon-oxidasa loda sintetizada por marinomonas mediterr\u00e1nea. papel de la flavoprote\u00edna lodb en la generaci\u00f3n del cofactor quin\u00f3nico de loda, mediante modificaci\u00f3n post-traduccional.<\/strong>\u00ab<\/h3>\n<ul>\n<li><strong>T\u00edtulo de la tesis:<\/strong>\u00a0 Estudio de la lisina-\u00e9psilon-oxidasa loda sintetizada por marinomonas mediterr\u00e1nea. papel de la flavoprote\u00edna lodb en la generaci\u00f3n del cofactor quin\u00f3nico de loda, mediante modificaci\u00f3n post-traduccional. <\/li>\n<li><strong>Autor:<\/strong>\u00a0 Mar\u00eda  Dolores Chacon Verdu <\/li>\n<li><strong>Universidad:<\/strong>\u00a0 Murcia<\/li>\n<li><strong>Fecha de lectura de la tesis:<\/strong>\u00a0 03\/07\/2015<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<h3>Direcci\u00f3n y tribunal<\/h3>\n<ul>\n<li><strong>Director de la tesis<\/strong>\n<ul>\n<li>Antonio Sanchez Amat<\/li>\n<\/ul>\n<\/li>\n<li><strong>Tribunal<\/strong>\n<ul>\n<li>Presidente del tribunal: manuel Canovas diaz <\/li>\n<li>subramanian Padmanabhan iyer (vocal)<\/li>\n<li>Francisco ignacio Javier Pastor blasco (vocal)<\/li>\n<li>Manuel Ferrer Martinez (vocal)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Tesis doctoral de Mar\u00eda Dolores Chacon Verdu Las l-amino\u00e1cido oxidasas (laos) son enzimas que oxidan amino\u00e1cidos generando el ceto\u00e1cido correspondiente, [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"site-sidebar-layout":"default","site-content-layout":"","ast-site-content-layout":"","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"","ast-breadcrumbs-content":"","ast-featured-img":"","footer-sml-layout":"","theme-transparent-header-meta":"","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"default","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center 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