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Cómo una mutación de un solo punto en la peroxidasa del rábano picante mejora notablemente la enantioselectividad
Eugene Antipov1, Art E Cho, Alexander M Klibanov
1Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Journal of the American Chemical Society
|July 21, 2009
Resumen
La mayoría de los mutantes de la peroxidasa (HRP) del rábano picante muestran enantioselectividad, a diferencia del tipo silvestre. La variante Arg178Glu mostró una preferencia significativa de 25 veces para el (S) -enantiómero de los fenoles quirales.
Área de la Ciencia:
- La biocatálisis por biocatálisis.
- Ingeniería de enzimas Ingeniería de enzimas Ingeniería de enzimas
- Química orgánica es la química orgánica.
Sus antecedentes:
- La peroxidasa del rábano picante (HRP) es una enzima ampliamente estudiada.
- La enantioselectividad en las reacciones enzimáticas es crucial para la síntesis de compuestos quirales.
- La modificación de los sitios activos de la enzima puede alterar la especificidad del sustrato y la enantioselectividad.
Objetivo del estudio:
- Para investigar el impacto de las mutaciones en la posición 178 de HRP en su enantioselectividad.
- Para identificar variantes de HRP con una mayor enantioselectividad hacia los fenoles quirales.
- Para dilucidar el mecanismo detrás de los cambios de enantioselectividad observados.
Principales métodos:
- La mutagénesis dirigida al sitio se utilizó para crear variantes de HRP con mutaciones en la posición 178.
- Se realizaron ensayos de oxidación enzimática utilizando varios sustratos quirales de fenol.
- El análisis cinético y el modelado molecular se emplearon para estudiar las interacciones enzima-sustrato y los estados de transición.
Principales resultados:
- La mayoría de los mutantes de HRP exhibieron enantioselectividad, a diferencia de la enzima de tipo salvaje.
- La variante Arg178Glu mostró la enantioselectividad más alta, con una preferencia de 25 veces para el (S) -enantiómero.
- El modelado molecular indicó que la repulsión electrostática entre Glu178 y el estado de transición del (R) -enantiómero contribuye a la enantioselectividad.
Conclusiones:
- Las mutaciones en la posición 178 de HRP pueden mejorar significativamente la enantioselectividad hacia los fenoles quirales.
- La mutación Arg178Glu representa una variante prometedora para la síntesis asimétrica.
- Comprender el papel de las interacciones electrostáticas proporciona información para el diseño racional de enzimas.
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