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Mejorar la actividad de una enzima natural mediante la incorporación de aminoácidos no naturales
Isaac N Ugwumba1, Kiyoshi Ozawa, Zhi-Qiang Xu
1Commonwealth Scientific and Industrial Research Organization, Black Mountain, Canberra, Australia.
Journal of the American Chemical Society
|December 18, 2010
Resumen
Las fosfotriesterasas bacterianas diseñadas con aminoácidos no naturales muestran un aumento de 8 a 11 veces en la hidrólisis de pesticidas. Esto demuestra que los aminoácidos diseñados pueden expandir la función enzimática y la evolución más allá de los límites naturales.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Ingeniería de enzimas Ingeniería de enzimas Ingeniería de enzimas
- Biología sintética Biología sintética.
Sus antecedentes:
- Las fosfotriesterasas bacterianas son altamente eficientes en la hidrólisis del pesticida paraoxón.
- Se cree que su límite evolutivo natural para la actividad de hidrólisis del paraoxón está cerca de .
- Los residuos del sitio activo periférico juegan un papel en las etapas catalíticas no químicas.
Objetivo del estudio:
- Investigar si los aminoácidos no naturales pueden mejorar la tasa de rotación catalítica de las fosfotriesterasas.
- Para investigar el papel de los residuos del sitio activo periférico en la unión al sustrato y la liberación del producto.
- Para diseñar una mayor actividad de hidrólisis de plaguicidas en las fosfotriesterasas bacterianas.
Principales métodos:
- La tirosina sustituida en la posición 309 por aminoácidos no naturales: L-(7-hidroxicumarina-4-il) etilglicina (Hco) y L-(7-metilcumarina-4-il) etilglicina.
- Incluidos los aminoácidos naturales leucina, fenilalanina y triptófano para la comparación.
- Se realizó un análisis cinético para evaluar la eficiencia catalítica e identificar los pasos que limitan la velocidad.
Principales resultados:
- El aminoácido Hco no natural aumentó significativamente el paso de liberación del producto que limita la velocidad.
- El grupo 7-hidroxilo de Hco, cuando se desprotona, repele electrostáticamente el producto cargado negativamente.
- Logró una mejora de 8 a 11 veces en la actividad catalítica con una sola mutación diseñada racionalmente.
- Esta mejora supera los esfuerzos realizados utilizando mutaciones naturales de aminoácidos.
Conclusiones:
- Los aminoácidos no naturales ofrecen una herramienta poderosa para mejorar las funciones enzimáticas existentes.
- Los aminoácidos diseñadores proporcionan acceso a nuevas secuencias y espacios funcionales para la evolución de las enzimas.
- Este enfoque demuestra un potencial significativo para la ingeniería de enzimas más allá de sus capacidades naturales.
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