Publication: Estudio cristalográfico de los mecanismos de especificidad y diseño molecular de glicosidasas para la producción de oligosacáridos prebióticos y derivados bioactivos
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2018-08-23
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Universidad Complutense de Madrid
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Abstract
Según diversos estudios epidemiológicos, la alimentación juega un papel determinante en la salud humana. Se ha demostrado que la dieta tiene un efecto directo sobre la composición y el equilibrio de la microbiota intestinal y, ésta, sobre la salud del individuo. Por ello, se buscan nuevos alimentos “funcionales”, como los oligosacáridos prebióticos, que son capaces de estimular el crecimiento de bacterias beneficiosas. Por otra parte, la glicosilación de distintos compuestos químicos mejora sus propiedades y su biodisponibilidad. Estos nuevos compuestos bioactivos aún no están tan desarrollados como los prebióticos, pero tienen una gran proyección de futuro en la industria farmacéutica. La investigación sobre la biosíntesis de estos productos conlleva tanto el estudio de nuevos compuestos con mejores propiedades funcionales como la búsqueda de formas más eficaces de producirlos. En este aspecto, la Biotecnología Enzimática se ha impuesto frente a otras metodologías de química tradicional gracias a su excelente regio-‐, quimio-‐ y estéreo-‐especificidad. Y para profundizar en esta metodología, la Cristalografía de rayos X genera la información estructural básica para el diseño racional en la ingeniería de proteínas. Por todo ello, esta tesis se ha centrado en el estudio cristalográfico de glicosil hidrolasas (GH) que son las enzimas capaces de metabolizar carbohidratos, los biocompuestos estructuralmente más diversos en la naturaleza. Estas enzimas, altamente específicas y eficientes, intervienen en multitud de procesos biológicos esenciales por lo que un conocimiento detallado de su función a nivel molecular es clave para entender dichos procesos y controlar numerosas enfermedades, además de permitir optimizar su aplicación biotecnológica. En este manuscrito, se presentan cuatro trabajos de investigación realizados con enzimas productoras de carbohidratos prebióticos del tipo FOS (fructooligosacáridos), IMOS (isomaltooligosacáridos) o β-‐glucanos, además de explorar su capacidad para sintetizar otros compuestos bioactivos. El objetivo general de la investigación es avanzar en el esclarecimiento de los mecanismos de reconocimiento proteína-‐carbohidrato que modulan la especificidad de glicosidasas, mediante la Cristalografía de proteínas. Además de las implicaciones fundamentales para entender los procesos en los que estas enzimas intervienen, este conocimiento estructural es la base fundamental para realizar la ingeniería molecular de enzimas. El fin último es producir oligosacáridos con propiedades prebióticas, diseñados específicamente para manipular la microbiota intestinal del individuo, lo que abre la puerta a una futura medicina preventiva personalizada...
Several epidemiological studies have shown that food plays a key role in human health. It has been shown that diet has a direct effect on the composition and balance of the intestinal microbiota and this, in turn, on health. Therefore, new "functional" foods are sought, many of which are prebiotic oligosaccharides that selectively stimulate the growth of beneficial bacteria. On the other hand, the glycosylation of different chemical compounds improves their properties and their bioavailability. These new bioactive compounds have yet to be fully exploited, being increasingly demanded by the pharmaceutical industry. Research on biosynthesis of these products involves the study of new compounds with better functional properties and, also, the development of more efficient production methods. Thus, Enzymatic Biotechnology has overcome other traditional chemical methodologies, due to its excellent regio-‐, chemo-‐ and stereo-‐specificity. Furthermore, X-‐ray crystallography generates the basic structural information for rational design and protein engineering. Accordingly, this thesis has focused on the crystallographic study of glycosyl hydrolases (GH), enzymes that metabolize carbohydrates, the most structurally diverse biocompounds in nature. These highly specific and efficient enzymes are involved in many essential biological processes. A detailed knowledge of their molecular function is crucial to understand these processes and control numerous diseases, and is necessary to optimize their biotechnological application. In this manuscript, we present the research work performed on four enzymes that can generate prebiotics of the types FOS (fructooligosaccharides), IMOS (isomaltooligosaccharides) or β-‐glucans. In addition, we have explored their ability to synthesize other bioactive compounds. The general goal of the work is to shed light into the intricate mechanisms of protein-‐ carbohydrate recognition that modulate the specificity of glycosidases, through protein crystallography. In addition to the fundamental implications for understanding the processes in which these enzymes are involved, this structural knowledge is crucial for performing the molecular engineering of enzymes. The ultimate goal is to produce oligosaccharides with prebiotic properties, specifically designed to manipulate the individual's intestinal microbiota, opening the door to personalized preventive medicine...
Several epidemiological studies have shown that food plays a key role in human health. It has been shown that diet has a direct effect on the composition and balance of the intestinal microbiota and this, in turn, on health. Therefore, new "functional" foods are sought, many of which are prebiotic oligosaccharides that selectively stimulate the growth of beneficial bacteria. On the other hand, the glycosylation of different chemical compounds improves their properties and their bioavailability. These new bioactive compounds have yet to be fully exploited, being increasingly demanded by the pharmaceutical industry. Research on biosynthesis of these products involves the study of new compounds with better functional properties and, also, the development of more efficient production methods. Thus, Enzymatic Biotechnology has overcome other traditional chemical methodologies, due to its excellent regio-‐, chemo-‐ and stereo-‐specificity. Furthermore, X-‐ray crystallography generates the basic structural information for rational design and protein engineering. Accordingly, this thesis has focused on the crystallographic study of glycosyl hydrolases (GH), enzymes that metabolize carbohydrates, the most structurally diverse biocompounds in nature. These highly specific and efficient enzymes are involved in many essential biological processes. A detailed knowledge of their molecular function is crucial to understand these processes and control numerous diseases, and is necessary to optimize their biotechnological application. In this manuscript, we present the research work performed on four enzymes that can generate prebiotics of the types FOS (fructooligosaccharides), IMOS (isomaltooligosaccharides) or β-‐glucans. In addition, we have explored their ability to synthesize other bioactive compounds. The general goal of the work is to shed light into the intricate mechanisms of protein-‐ carbohydrate recognition that modulate the specificity of glycosidases, through protein crystallography. In addition to the fundamental implications for understanding the processes in which these enzymes are involved, this structural knowledge is crucial for performing the molecular engineering of enzymes. The ultimate goal is to produce oligosaccharides with prebiotic properties, specifically designed to manipulate the individual's intestinal microbiota, opening the door to personalized preventive medicine...
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Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Bioquímica y Biología Molecular I, leída el 10-02-2017