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A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
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Cómo la evolución dirigida remodela el paisaje energético en una enzima para impulsar la catálisis
Renee Otten1, Ricardo A P Pádua1, H Adrian Bunzel2
1Howard Hughes Medical Institute and Department of Biochemistry, Brandeis University, Waltham, MA 02454, USA.
Science (New York, N.Y.)
|November 20, 2020
Resumen
La evolución dirigida optimizó una enzima diseñada por computación para la transferencia de protones, aumentando drásticamente su velocidad catalítica. Este proceso refinó el andamio de proteínas
Área de la Ciencia:
- Biocatálisis y ingeniería enzimática
- El diseño y la evolución de las proteínas
- Química computacional y modelado molecular
Sus antecedentes:
- Los biocatalizadores ofrecen potencial para nuevas transformaciones químicas.
- El diseño computacional y la evolución en laboratorio son herramientas poderosas para la optimización de enzimas.
- La comprensión de la dinámica conformacional de la enzima es clave para mejorar la eficiencia catalítica.
Objetivo del estudio:
- Investigar estrategias moleculares para aumentar la función catalítica utilizando biocatalizadores diseñados.
- Para aclarar cómo la evolución dirigida altera la dinámica del andamio de proteínas para mejorar el rendimiento de las enzimas.
- Comprender el papel de los conjuntos conformacionales en la catálisis enzimática.
Principales métodos:
- Diseño computacional de las enzimas.
- La evolución dirigida por el laboratorio.
- Espectroscopia de resonancia magnética nuclear (RMN).
- Es una cristalografía de rayos X.
- Mediciones de la cinética del flujo detenido.
Principales resultados:
- La evolución dirigida aceleró la reacción de transferencia de protones de una enzima diseñada en casi nueve órdenes de magnitud.
- La optimización condujo a un conjunto conformacional estrecho y altamente activo del andamio de proteínas.
- Las mutaciones adquiridas indujeron cambios conformacionales globales y reordenamientos de la columna vertebral.
- Estos cambios organizaron cooperativamente los residuos catalíticos y estabilizaron el estado de transición.
Conclusiones:
- El muestreo explícito y la estabilización de los subestados conformacionales productivos pueden facilitar el desarrollo de nuevos catalizadores de proteínas.
- La evolución dirigida refina la dinámica conformacional de las proteínas para mejorar la función catalítica.
- La estabilización del estado de transición es crítica para lograr una alta actividad enzimática.
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