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La ciclización de péptidos catalizada por el dominio tioesterasa de la tirocidina sintetasa

J W Trauger¹, R M Kohli, H D Mootz

¹Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.

|September 23, 2000

Resumen

Este estudio demuestra que los dominios extirpados de tioesterasa (TE) pueden catalizar de forma independiente la macrociclado de péptido-tioésteres. Este hallazgo es crucial para la comprensión de la especificidad del sustrato TE y la ingeniería de nuevas biosíntesis de productos naturales.

Área de la Ciencia:

La bioquímica es la bioquímica.
Biología Molecular Biología Molecular
Biosíntesis de Productos Naturales Biosíntesis de Productos Naturales

Sus antecedentes:

Las megasintasas de múltiples dominios sintetizan productos naturales macrocíclicos, con dominios carboxiterminales de tioesterasa (TE) que median la ciclización y la liberación.
La capacidad catalítica independiente y la especificidad del sustrato de los dominios TE han permanecido en gran medida indeterminadas debido a su integración dentro de complejos enzimáticos más grandes.

Objetivo del estudio:

Investigar si los dominios extirpados de tioesterasa (TE) pueden catalizar la macrociclado independientemente de los dominios enzimáticos aguas arriba.
Determinar la especificidad del sustrato del dominio TE de la tirocidina sintetasa para aplicaciones potenciales en la biosíntesis de ingeniería.

Principales métodos:

Excisión del dominio TE de la tirocidina sintetasa.
Evaluación de la actividad catalítica del dominio TE aislado en decapeptido-tioésteres ciclizantes y pentapeptido-tioésteres dimerizantes/ciclizantes.
Variación sistemática de sustratos peptídico-tioéster para analizar las tasas de ciclización e identificar los residuos críticos.

Principales resultados:

El dominio TE extirpado catalizó de manera eficiente la ciclización de un teoestero de decapeptido para formar tirrocidina A.
El dominio TE también catalizó la dimerización y posterior ciclización de un pentapeptido-tioéster para formar gramicidina S.
El análisis reveló que solo dos residuos específicos dentro del sustrato del decapeptido son críticos para una ciclización eficiente.

Conclusiones:

Los dominios de TE extirpados poseen una actividad catalítica intrínseca para la macrociclado, independiente de la maquinaria enzimática aguas arriba.
El perfil de especificidad de sustrato identificado sugiere una amplia aplicabilidad de los dominios TE para ciclizar diversos sustratos en la biosíntesis de ingeniería.
Este trabajo proporciona una base para el diseño de nuevos péptidos cíclicos y productos naturales a través de enfoques de biología sintética.

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