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La estereospecificidad del dominio de la deshidratasa de la eritromicina policetida sintasa

Chiara R Valenzano¹, Young-Ok You, Ashish Garg

¹Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912-9108, USA.

Journal of the American Chemical Society

|October 8, 2010

Resumen

El dominio deshidratasa de la 6-deoxyeritronolide B sintasa (DEBS DH4) cataliza exclusivamente la síntesis, la eliminación y la adición de agua. Esta enzima es la enzima

Área de la Ciencia:

Bioquímica y Enzimología.
Síntesis de Polyketide Síntesis de Polyketide también se conoce como Polyketide.
Química orgánica es la química orgánica.

Sus antecedentes:

El dominio de la deshidratasa (DH) es un componente clave de las poliquetidas sintasas modulares (PKS).
Comprender el curso estereoquímico de las reacciones de deshidratación es crucial para elucidar las líneas de ensamblaje de PKS.

Objetivo del estudio:

Investigar la especificidad del sustrato y el resultado estereocímico del dominio de la deshidratasa (DH4) del módulo 4 de la 6-deoxyeritronolide B sintasa (DEBS).
Para caracterizar las reacciones de hidratación y deshidratación catalizadas por enzimas que involucran intermediarios específicos de proteínas portadoras de acilo (ACP).

Principales métodos:

Incubación de DEBS DH4 recombinante con intermediarios acil-ACP preparados quimioenzimáticamente o generados sintéticamente.
Generación in situ de productos intermedios utilizando combinaciones de DEBS y otros dominios de sintasa (por ejemplo, TYLS KR1, RIFS KR7).
Análisis de los productos de reacción utilizando la espectrometría de masas por cromatografía de gases (GC-MS) y la espectrometría de masas por cromatografía de líquidos (LC-MS).

Principales resultados:

DEBS DH4 cataliza exclusivamente una eliminación síncrona de agua de anti-(2R,3R)-2-metil-3-hidroxipentanilo-ACP (2a-ACP) para obtener 3-ACP.
La reacción de hidratación inversa por DH4 da como resultado una mezcla de equilibrio de 2a-ACP y 3-ACP.
DH4 demuestra estricta estereospecificidad, deshidratando sólo sustratos específicos de anti- y sin-configurados hidroxilo-ACP, y fue utilizado para sintetizar un complejo derivado heptenoyl-ACP.

Conclusiones:

El dominio de la deshidratasa del módulo 4 de DEBS opera con exclusiva sin estereoquímica para la eliminación y adición de agua.
Este control estereoquímico es crítico para el ensamblaje preciso de la columna vertebral poliquetídica en DEBS.
Los hallazgos proporcionan información sobre el reconocimiento del sustrato y el mecanismo catalítico de los dominios DH en PKS.

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