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Marinomonas Mediterranea Perteneciente a Una Nueva Familia De Quinoproteínas

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Marinomonas Mediterranea Perteneciente a Una Nueva Familia De Quinoproteínas UNIVERSIDAD DE MURCIA FACULTAD DE BIOLOGÍA Identificación y Caracterización de una Glicina Oxidasa en Marinomonas mediterranea Perteneciente a una Nueva Familia de Quinoproteínas D. Jonatan Cristian Campillo Brocal 2016 UNIVERSIDAD DE MURCIA FACULTAD DE BIOLOGÍA Identificación y caracterización de una glicina oxidasa en Marinomonas mediterranea perteneciente a una nueva familia de quinoproteínas D. Jonatan Cristian Campillo Brocal 2016 El presente trabajo ha sido entregado para optar al grado de doctor en "Biología Molecular y Biotecnología". Asimismo ha sido propuesto para Mención de Doctorado Internacional en virtud a la estancia predoctoral realizada en el “Laboratorio de Biodiversidad Microbiana y Biotecnológica” (LBBM) situado en el Observatorio Oceanográfico de Banyuls sur Mer (Francia). Esta Tesis Doctoral ha sido desarrollada durante el periodo de disfrute de una beca-contrato predoctoral de la Universidad de Murcia y ha sido financiada por el Ministerio de Ciencia e Innovación (Proyecto BIO2010-15226) y por la Fundación Séneca de la Región de Murcia (Proyecto 11867/PI/09). El trabajo realizado en la presente memoria ha dado lugar a las siguientes publicaciones: 1. Campillo-Brocal, J. C., P. Lucas-Elío and A. Sánchez-Amat. (2013) Identification in Marinomonas mediterranea of a novel quinoprotein with glycine oxidase activity. MicrobiologyOpen. 2 (4): 684-694. doi: 10.1002/mbo3.107. 2. Chacón-Verdú, M. D., J. C. Campillo-Brocal, P. Lucas-Elío, V. L. Davidson and A. Sánchez-Amat. (2015) Characterization of recombinant biosynthetic precursors of the cysteine tryptophylquinone cofactors of L-lysine-epsilon-oxidase and glycine oxidase from Marinomonas mediterranea. BBA-Proteins and Proteomics. 1854 (9): 1123–1131. doi: 10.1016/j.bbapap.2014.12.018. 3. Campillo-Brocal, J. C., M. D. Chacón-Verdú, P. Lucas-Elío and A. Sánchez- Amat. (2015) Distribution in microbial genomes of genes similar to lodA and goxA which encode a novel family of quinoproteins with amino acid oxidase activity. BMC Genomics. 16:231. doi: 10.1186/s12864-015-1455-y. 4. Campillo-Brocal, J. C., P. Lucas-Elío and A. Sánchez-Amat. (2015) Distribution in different organisms of amino acid oxidases with FAD or a quinone as cofactor and their role as antimicrobial proteins in marine bacteria. Marine Drugs. 13 (12): 7403-7418. Review. doi: 10.3390/md13127073. 5. Sehanobish, E., J. C. Campillo-Brocal, H. R. Williamson, A. Sánchez-Amat and V. L. Davidson. (2016) Interaction of GoxA with its modifying enzyme and its subunit assembly are dependent on the extent of cysteine tryptophylquinone biosynthesis. Biochemistry. 55 (16): 2305–2308. doi: 10.1021/acs.biochem.6b00274. Además ha sido expuesto en los siguientes Congresos nacionales e internacionales: Gordon Research Conference on Protein Cofactors, Radicals & Quinones (2012). Mount Holyoke College South Hadley, MA (EEUU). OxiZymes 2012. Marsella (Francia). VII Congreso Internacional de la Federación Española de Biotecnólogos (BAC2013). Sevilla (España). XIV International Symposium VII European Conference on Marine Natural Products (MaNaPro) (2013). La Toja (España). I Congreso Internacional Campus Mare Nostrum de Jóvenes Investigadores en el Mediterráneo (2013). Murcia (España). The Aragolab Students Meeting (Journée Aragoyenne) (2014). Banyuls sur Mer (Francia). OxiZymes 2014. Viena (Austria). The Fourth International Conference on Cofactors (ICC-04) (2014). Parma (Italia). OxiZymes 2016. Wageningen (Holanda). AGRADECIMIENTOS En las siguientes líneas tengo la intención de mostrar mi gratitud por la ayuda recibida a lo largo de estos años de Tesis Doctoral, enfrentándome a la ardua tarea de no olvidar ningún nombre, de ser breve y de no caer excesivamente en el sentimentalismo y la cursilería. De antemano pido disculpas por no ser capaz de expresar lo agradecido que estoy y lo mucho que les debo a esas personas que han contribuido a la conquista de este sueño. En primer lugar, como no podía ser de otra forma quisiera dar las gracias a mis directores de Tesis y mentores, el Dr. Antonio Sánchez Amat y la Dra. Patricia Elío Lucas. Con Patricia descubrí la Microbiología allá por mi segundo año de carrera. Muchas gracias por ello y por tu disposición, generosidad, positividad y porque contigo di mis primeros pasos en un laboratorio. Antonio hizo que quisiera adentrarme aún más en este microscópico mundo y despertó mi faceta como investigador. Gracias por tu apoyo y enseñanzas, y por transmitir tu pasión por la ciencia y la investigación día tras día. Ambos me habéis enseñado prácticamente todo lo que se científicamente hablando y por ello os estaré eternamente agradecido. Agradecer al profesor Marcelino Suzuki, por darme la posibilidad de realizar una estancia en su laboratorio y por hacerme de vez en cuando un hueco en su complicada agenda. En general, agradecer la enorme hospitalidad y ayuda recibida durante esta estancia por la gente del Observatorio Oceanográfico de Banyuls sur Mer (Francia) que me permitió crecer como científico y como persona: Luis, Méryl, Sandra, Mehdi y en especial a Laurent y a Sonja, por estar siempre dispuestos a echarme una mano tanto dentro como fuera del laboratorio. Al personal del Servicio de Biología Molecular del SAI de la Universidad de Murcia, principalmente al Dr. Alejandro Torrecillas por la espectrometría de masas y al Dr. Cesar Flores por la secuenciación de ADN. Por vuestro eficiente y gran trabajo. A todos los profesores e investigadores del dpto. de Genética y Microbiología por vuestro apoyo en algún u otro momento a lo largo de estos años. Especialmente al profesor de profesores, Francisco Torrella, por su sabiduría, sentido del humor y por transmitir tan efusivamente su inquietud científica. A Alejandro, siempre tan servicial. A Luis Miguel y Paco por nuestras acaloradas charlas sobre fútbol y deportes varios. A los compañeros de facultad y laboratorio con los que he tenido la suerte de coincidir, por todos los momentos que hemos vivido juntos incluyendo experimentos, comidas, autoclaves, festejos varios, etc. A Dani, Luisa, Sandra, Aida, Mari Carmen y en especial a Rafa y Mariola con los que he compartido la mayor parte de este camino. Porque nadie mejor que vosotros sabe lo que se sufre y disfruta haciendo el Doctorado. Sin olvidar al “chico del pollo” Fabio, ese “cazzo” de italiano que me aportó un soplo de aire fresco durante su corta estancia en Murcia. También a mis amigos del dpto. de Fisio Animal, María, Bea, Antonio y en especial a Domingo, un auténtico figura. Una mención distinguida para mis “biofriends”, con los que inicié mi andadura por la facultad de Biología. Al principio sólo eran compañeros de carrera, pero poco a poco se fueron convirtiendo en los mejores amigos que uno puede desear: Ana, Débora, Antonio, Edu, Jesús, Vero, Mari Paz, Juanjo, Rafa, Pablo y Pedro. He dejado para el final y no por ello menos importante a mi familia. Aprovecho la ocasión para expresar, ya que no lo hago muy a menudo, lo realmente afortunado que me siento de teneros. Aquí puedo incluir a los miembros del “Ekipo” con los que he vivido tanto que han pasado a ser parte de mi familia. A la familia Algarra-Oñate, por ser mi segunda familia. A mis primos, tíos y sobre todo mis abuelos. A mis hermanos Santi e Indira, y a mis padres Santiago y Teresa, porque les debo todo lo que soy. Por enseñarme que los estudios eran lo primero y por inculcarme que sin esfuerzo y sacrificio no hay recompensa. Cuanto me enorgullece compartir las ganas de seguir formulando preguntas y de seguir aprendiendo día tras día. Por último, especialmente gracias a ti, Blanca, porque nadie como tú sabe bien lo que significa para mi poder estar hoy escribiendo estos agradecimientos y culminando este triunfo. Por ser compañera de vida, por darle sentido a mi mundo y porque sin tu apoyo y amor incondicional no lo habría conseguido. Por todo y por mucho más, ¡gracias! “Según vamos adquiriendo conocimiento, las cosas no se hacen más comprensibles sino más misteriosas” Albert Schweitzer Para mi familia y amigos, para M. mediterranea, para quien sostiene este libro, y en especial a ti, Blanca ABREVIATURAS aa: aminoácido Gox: glicina oxidasa Amp: ampicilina GoxA: glicina oxidasa de Marinomonas amu: unidad de masa atómica mediterranea (producto de AO: aminoácido oxidasa Marme_1655) AP: presión atmosférica HPLC: cromatografía líquida de alta Ara: arabinosa resolución ATCC: Colección Americana de HRP: peroxidasa de rábano Cultivos Tipo IMG: Integrated Microbial Genomes BLAST: herramienta bioinformática de (DOE-JGI, USA) alineamiento de secuencias de tipo local IPTG: isopropil-beta-D- BSA: albúmina de suero bovino tiogalactopiranósido CECT: Colección Española de Cultivos JGI: Joint Genome Institute (USA) Tipo Kb: kilobases de DNA o 1000 pb Cm: cloranfenicol kDa: kilodalton CTQ: cisteína triptofilquinona Km: kanamicina DAO: D-aminoácido oxidasa LAO: L-aminoácido oxidasa DBM: motivo de unión a dinucleótidos LOD: actividad lisina oxidasa DMSO: dimetil sulfóxido LodA: L-lisina épsilon-oxidasa DNA: ácido desoxirribonucleico (producto de Marme_2662) DO600: densidad óptica medida a 600nm LTQ: lisina tirosilquinona Dopa: 3,4-dihidroxifenilalanina m/v: relación masa/volumen DTT: ditiotreitol m/z: relación masa/carga EC: Comisión de Enzimas MADH: metilamina deshidrogenasa EDTA: etilén diamino tetracetato (sal MALDI: ionización por desorción láser disódica) asistida por una matriz ESI: ionización por electrospray mau: operón de utilización de EtOH: etanol metilamina FAD: flavín dinucleótido MCO: multicobreoxidasa FMN: flavín
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