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Evolución dirigida de la nueva actividad catalítica utilizando el andamio de barril alfa/beta
M M Altamirano1, J M Blackburn, C Aguayo
1Cambridge Centre for Protein Engineering, and Cambridge University Chemical Laboratory, MRC Centre, UK.
Nature
|February 25, 2000
Resumen
Los científicos desarrollaron una nueva enzima utilizando una estructura proteica común, el andamio de barril alfa / beta. Este enfoque de evolución dirigida creó una nueva isomerasa de fosforibosilantranilato con mayor especificidad, allanando el camino para nuevos biocatalizadores.
Área de la Ciencia:
- Biocatálisis y ingeniería de enzimas.
- Evolución del andamio de proteínas en la evolución del andamio de proteínas.
- Evolución dirigida de las enzimas.
Sus antecedentes:
- Las enzimas son biocatalizadores cruciales en los sistemas biológicos, pero las aplicaciones industriales requieren nuevas enzimas con funciones a medida.
- La evolución de las enzimas a menudo reutiliza los andamios proteicos existentes para adquirir nuevas actividades catalíticas.
- El barril alfa / beta es un pliegue de proteínas prevalente, lo que sugiere su potencial como un andamio versátil.
Objetivo del estudio:
- Para imitar la evolución natural de las enzimas mediante la ingeniería de nuevas funciones catalíticas en un andamio de proteínas existente.
- Para desarrollar la actividad de la isomerasa del fosforibosilantranilato utilizando el andamio alfa/beta de la indol-3-glicerol-fosfato sintasa.
- Demostrar la viabilidad de la evolución dirigida para crear nuevos biocatalizadores a partir de pliegues de proteínas comunes.
Principales métodos:
- Combinó un sitio de unión para el fosforibosilantranilato con una plantilla catalítica de isomerasa dentro del andamio de barril alfa/beta.
- Utilizado en la mutagénesis in vitro y la recombinación dirigida a la plantilla catalítica.
- Se emplea la selección in vivo para identificar y aislar enzimas evolucionadas.
Principales resultados:
- Evolucionó con éxito una nueva enzima con actividad de isomerasa de fosforibosilantranilato a partir del andamio de la indol-3-glicerol-fosfato sintasa.
- La enzima modificada exhibió propiedades catalíticas comparables a las de la enzima natural.
- La nueva enzima demostró una constante de especificidad aún más alta que su contraparte natural.
Conclusiones:
- El andamio de barril alfa/beta es una plataforma prometedora para la evolución dirigida de nuevos biocatalizadores.
- Este estudio demuestra la evolución divergente exitosa de la función enzimática utilizando un pliegue proteico común.
- Los hallazgos apoyan la estrategia de reutilizar los andamios de proteínas existentes para crear nuevas actividades enzimáticas.
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