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Formación estereospecífica de enlaces dobles E- y Z-disubstituidos por dominios de deshidrataza de los módulos 1 y 2

Dhara D Shah¹, Young-Ok You¹, David E Cane¹

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

Journal of the American Chemical Society

|September 14, 2017

Resumen

El dominio deshidratasa FosDH1 y FosDH2 de la fostriecina policetida sintasa (PKS) exhiben conversiones estereospecíficas de sustrato. Estas enzimas juegan un papel clave en la biosíntesis de policetidas complejas, demostrando distintas actividades catalíticas.

Área de la Ciencia:

La bioquímica
Enzimología
Biología molecular

Sus antecedentes:

La fostriecina policetida sintasa (PKS) es un mecanismo enzimático complejo responsable de la biosíntesis del potente agente anticancerígeno fostriecina.
Los dominios de deshidrataza (DH) y cetorreductaza (KR) dentro de PKS son cruciales para catalizar modificaciones estereospecíficas de las cadenas de policetida en crecimiento.
Comprender las funciones específicas y las especificidades del sustrato de dominios individuales como FosDH1 y FosDH2 es esencial para dilucidar las vías biosintéticas de la PKS.

Objetivo del estudio:

Caracterizar las actividades enzimáticas y las especificidades del sustrato de los dominios deshidratasa FosDH1 y FosDH2 de la fostriecina PKS.
Investigar los resultados estereoquímicos de las reacciones catalizadas por FosDH1 y FosDH2 utilizando sustratos específicos de hidroxibutirilo y butenoilo.
Explorar las funciones potenciales de estos dominios en el contexto de la vía biosintética general de la fostriecina.

Principales métodos:

Se realizaron ensayos enzimáticos utilizando dominios de deshidratasa purificados (FosDH1, FosDH2) y cetorreductaza (FosKR2) de la fostriecina PKS.
Se sintetizaron y utilizaron estereoisómeros de sustrato, incluidos (3R) - y (3S) - 3-hidroxibutirilo-FosACP y (E) - y (Z) - 2-butenoil-FosACP.
El análisis del producto se realizó mediante cromatografía líquida- espectrometría de masas en tándem (LC-MS/ MS) y cromatografía quiral de gases- espectrometría de masas (GC-MS) para la determinación estereoquímica.

Principales resultados:

FosDH1 demostró estricta estereospecificidad, catalizando la interconversión de (3R) -3-hidroxibutirilo-FosACP1 y (E) -2-butenoil-FosACP1, pero no sus contrapartes (3S) o (Z).
FosKR2 catalizó la reducción estereospecífica de 3-ketobutirilo-FosACP2 a (3S) -3-hidroxibutirilo-FosACP2, proporcionando un sustrato para FosDH2.
FosDH2 exhibió una aceptación más amplia del sustrato, catalizando las interconversiones que involucran sustratos de triketida y la hidratación estereospecífica de (Z) -2-butenoil-FosACP2 a (3S) -3-hidroxibutirilo-FosACP2. Inesperadamente, FosDH2 también se interconvirtió (3R) -3-hidroxibutirilo-FosACP2 y (E) -2-butenoil-FosACP2.

Conclusiones:

FosDH1 y FosDH2 poseen distintas especificidades de sustrato y preferencias estereoquímicas, lo que contribuye a la construcción precisa de la columna vertebral de la fostriecina policetida.
Los hallazgos proporcionan información sobre la lógica enzimática modular de la fostriecina PKS, destacando la acción coordinada de los dominios KR y DH.
La inesperada aceptación del sustrato de FosDH2 sugiere una posible flexibilidad en la función del dominio PKS o en rutas biosintéticas alternativas.

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