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Caracterización funcional de un dominio de deshidratasa de la picromicina poliquetida sintasa

Yang Li¹, Greg J Dodge², William D Fiers¹

¹†Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55455, United States.

Journal of the American Chemical Society

|June 2, 2015

Resumen

Este estudio caracteriza el dominio de la deshidratasa (DH) PikDH2 de las sintetasas policetidas (PKS), revelando su mecanismo para crear compuestos insaturados. Este trabajo permite el descubrimiento de nuevos productos naturales a través de la ingeniería metabólica.

Área de la Ciencia:

La bioquímica es la bioquímica.
Ingeniería Metabólica Ingeniería Metabólica.
Descubrimiento de productos naturales Descubrimiento de productos naturales.

Sus antecedentes:

Las vías de la poliquetida sintasa (PKS) son cruciales para la biosíntesis de productos naturales.
Los dominios de la deshidratasa (DH) dentro de las PKS son menos conocidos debido a las limitaciones de los ensayos.
La ingeniería metabólica de las vías de la PKS ofrece una ruta prometedora para el descubrimiento de nuevos productos naturales.

Objetivo del estudio:

Para realizar el primer análisis cinético de estado estacionario de un dominio PKS DH (PikDH2).
Para aclarar la especificidad del sustrato y el mecanismo catalítico de PikDH2.
Establecer una base para futuros estudios estructurales de los dominios PKS DH.

Principales métodos:

Se utilizó la cromatografía líquida-espectrometría de masas en tándem (LC-MS/MS) para la cuantificación del producto.
Se empleó una serie de sustratos sintéticos de triketida con enlaces de tioéter no hidrolizables.
Realizó mutagénesis dirigida al sitio, estudios de dependencia del pH y ensayos de inhibición basados en el mecanismo.

Principales resultados:

PikDH2 convierte los sustratos de d-alcohol en productos de trans-olefinas con cinética reversible (Keq = 1,22).
Se ha demostrado una estricta especificidad de sustrato, sin rotación para sustratos epímeros.
Identificó un grupo ionizable clave (pKa = 7.0) y caracterizó la inactivación irreversible por un inhibidor basado en el mecanismo.

Conclusiones:

PikDH2 es un dominio robusto y específico de PKS DH adecuado para la síntesis quimioenzimática preparatoria.
Las ideas cinéticas y mecanicistas sobre PikDH2 proporcionan una base para comprender la función PKS DH.
Este trabajo facilita la ingeniería de las vías PKS para el descubrimiento de nuevos productos naturales.

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