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Adaptabilidad estructural facilita la ligación del hemo de histidina en un citocromo P450

John A McIntosh¹, Thomas Heel¹, Andrew R Buller¹

¹Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology , 1200 East California Boulevard, Pasadena, California 91125, United States.

Journal of the American Chemical Society

|August 25, 2015

Resumen

Los investigadores exploraron los sitios activos de la enzima citocromo P450 (CYP) mediante la mutación de un residuo clave de cisteína en histidina. Este cambio estructural reveló nuevas actividades catalíticas y propiedades enzimáticas para futuras investigaciones.

Área de la Ciencia:

La bioquímica
Biología estructural
Enzimología

Sus antecedentes:

Las enzimas del citocromo P450 (CYP) son cruciales para el metabolismo, utilizando típicamente un residuo de cisteína conservado para ligar el hierro hemo.
Las mutaciones en esta cisteína pueden abolir la actividad natural de la monooxigenasa, pero pueden permitir nuevas reacciones de transferencia de carbenos y nitrenos.
Comprender la estructura del sitio activo del CYP es clave para diseñar nuevas funciones catalíticas.

Objetivo del estudio:

Para determinar la estructura cristalina de una enzima P450 ligada a la histidina.
Investigar las consecuencias estructurales y funcionales de la mutación de la cisteína axial en el CYP119.
Explorar el potencial de los sitios activos del CYP diseñados para la catálisis no natural.

Principales métodos:

Mutagénesis dirigida al sitio de la enzima CYP119 termostable de Sulfolobus acidocaldarius.
Cristalografía de rayos X para obtener la estructura de alta resolución de la variante T213A/C317H.
Análisis espectroscópico de varios mutantes de la cisteína axial.

Principales resultados:

Se determinó la primera estructura cristalina de un P450 ligado a la histidina (CYP119 T213A/ C317H), mostrando la coordinación del hierro hemo por la histidina.
La mutación en histidina indujo alteraciones significativas en la estructura general de la proteína.
Se encontró que la cisteína axial es reemplazable por otros aminoácidos sin comprometer el plegamiento de proteínas o la incorporación de hemo, lo que lleva a propiedades espectrales únicas.

Conclusiones:

El sitio activo del P450 ligado a la histidina es estructuralmente distinto y mantiene la coordinación del hemo.
Los sitios activos de CYP diseñados con diversas sustituciones de aminoácidos son estables y poseen propiedades únicas.
Estos hallazgos proporcionan una base para explorar nuevos mecanismos de catálisis y reacción mediados por P450.

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