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La formación del producto por la Promiscuous Lanthipeptide Synthetase ProcM está bajo control cinético

Yi Yu¹, Subha Mukherjee¹, Wilfred A van der Donk¹

¹†Department of Biochemistry, ‡Department of Chemistry, and §Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave. Urbana, Illinois 61801, United States.

Journal of the American Chemical Society

|March 25, 2015

Resumen

La lanthipeptida sintetasa ProcM exhibe una alta selectividad del producto, con mutaciones que afectan las tasas de ciclización y la regioselectividad. La formación inicial del anillo dicta la topología del producto final, no el control termodinámico.

Área de la Ciencia:

La bioquímica es la bioquímica.
Biología Molecular Biología Molecular
Productos naturales Química Productos naturales Química

Sus antecedentes:

Los latipeptidos son péptidos sintetizados por ribosomas y modificados post-traducionalmente (RiPP) con diversas actividades biológicas.
Las principales características estructurales incluyen puentes de lantionina (Lan) o metilantionina (MeLan).
La lanthipeptida sintetasa ProcM demuestra una amplia tolerancia al sustrato pero una alta selectividad del producto.

Objetivo del estudio:

Investigar el mecanismo de la regioselectividad en la síntesis de lanthipeptidos por ProcM.
Determinar el papel de los residuos del sitio activo y los eventos iniciales de ciclización en la formación de productos.
Explorar la influencia de las mutaciones en las tasas de ciclización y la topología del producto.

Principales métodos:

Mutagénesis dirigida al sitio de los residuos del sitio activo de ProcM (ligandos de zinc).
Análisis de las tasas de ciclización y la formación de productos utilizando sustratos lantipeptídicos específicos (ProcA1.1, ProcA2.8, ProcA3.3).
Caracterización de los productos de reacción para evaluar la regioselectividad y la topología de anillos.

Principales resultados:

Las mutaciones en los ligandos de zinc del sitio activo disminuyeron las tasas de ciclización, particularmente para el segundo paso de ciclización.
Mutaciones específicas alteraron la regioselectividad de la ciclización para el sustrato ProcA3.3, produciendo un nuevo producto principal.
ProcM no pudo corregir los intermediarios ciclados incorrectamente, lo que indica una falta de control termodinámico.

Conclusiones:

La alta regioselectividad de la formación de lanthipeptidos por ProcM está determinada principalmente por la selectividad de la formación inicial de anillos.
Las mutaciones que afectan al sitio activo influyen tanto en la cinética de la reacción como en la especificidad del producto.
La biosíntesis de Lanthipeptide está controlada cinéticamente, con eventos tempranos de ciclización que dirigen la estructura general del producto.

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