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Evolución en el laboratorio de las monooxigenasas Baeyer-Villiger robustas y enantioselectivas para la catálisis
1Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mulheim an der Ruhr, Germany. reetz@mpi-muelheim.mpg.de
Journal of the American Chemical Society
|October 8, 2009
Resumen
Los investigadores diseñaron la enzima fenilacetona monooxigenasa (PAMO) termostable para ampliar el alcance de su sustrato. Los nuevos mutantes exhiben una alta actividad y enantioselectividad para desafiar las resoluciones de cetonas, manteniendo la estabilidad de la enzima madre.
Área de la Ciencia:
- Biocatálisis e Ingeniería de Enzimas.
- Química orgánica es la química orgánica.
- Ingeniería de proteínas Ingeniería de proteínas.
Sus antecedentes:
- La fenilacetona monooxigenasa (PAMO) es una enzima termostable ideal para las reacciones enantioselectivas de Baeyer-Villiger.
- El PAMO de tipo silvestre exhibe un alcance de sustrato limitado, aceptando principalmente fenilacetona y análogos similares.
- Los esfuerzos previos de ingeniería de proteínas para expandir la gama de sustratos y la enantioselectividad de PAMO han tenido un éxito limitado.
Objetivo del estudio:
- Desarrollar nuevos mutantes PAMO con mayor actividad y enantioselectividad para una gama más amplia de sustratos cetónicos.
- Investigar una nueva estrategia de evolución de laboratorio dirigida a regiones específicas de proteínas para mejorar la función enzimática.
- Para mantener la inherente termostabilidad del tipo silvestre PAMO en las variantes de ingeniería.
Principales métodos:
- Empleó un enfoque bioinformático utilizando alineación de secuencias de ocho monooxigenasas de Baeyer-Villiger.
- Datos de estructura de rayos X integrados de PAMO y acoplamiento de ajuste inducido para identificar posibles sitios de aleatorización.
- Generación de biblioteca enfocada dirigida a residuos de prolina conservados en una región específica del bucle de la enzima.
Principales resultados:
- Se han desarrollado mutantes de PAMO que demuestran una actividad significativamente mayor y una enantioselectividad para la resolución de varias 2-arilo y 2-alquilociclohexanonas.
- Los mutantes diseñados procesaron con éxito cetonas bicíclicas estructuralmente distintas, expandiendo la aceptación del sustrato más allá del PAMO de tipo salvaje.
- Los mutantes más efectivos mantuvieron la excepcional termostabilidad característica del tipo salvaje padre PAMO.
Conclusiones:
- La nueva estrategia de evolución de laboratorio dirigida a los sitios de prolina conservados es altamente efectiva para mejorar el rendimiento catalítico de PAMO.
- Las variantes de PAMO diseñadas ofrecen biocatalizadores prometedores para las oxidaciones enantioselectivas de Baeyer-Villiger de diversos sustratos cetónicos.
- La "hipótesis de la prolina" justifica una mayor investigación en futuros estudios de evolución de enzimas para esta clase de enzimas.
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