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Diseño y evolución de una nueva actividad catalítica con un andamio proteico existente

Hee-Sung Park¹, Sung-Hun Nam, Jin Kak Lee

¹Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Kusung-Dong, Yusung-Gu, Daejon 305-701, Korea.

Science (New York, N.Y.)

|January 28, 2006

Resumen

La ingeniería de proteínas introdujo con éxito la actividad de la beta-lactamasa en un nuevo andamio enzimático. Esta enzima evolucionada hidroliza eficientemente la cefotaxima, mejorando la resistencia bacteriana.

Área de la Ciencia:

Ingeniería de proteínas Ingeniería de proteínas.
Diseño de la Enzima Diseño de la Enzima
La biocatálisis por biocatálisis.

Sus antecedentes:

El diseño de enzimas con nuevas funciones es un objetivo clave en la ingeniería de proteínas.
La modificación de los andamios proteicos existentes ofrece una estrategia prometedora para crear nuevos biocatalizadores.

Objetivo del estudio:

Desarrollar un método para alterar la actividad catalítica de la enzima mediante la modificación de los bucles del sitio activo.
Para introducir la actividad de la beta-lactamasa en el andamio de la glyoxalasa II.

Principales métodos:

Inserción, eliminación y sustitución simultáneas de bucles de sitio activo.
Actividad enzimática de ajuste fino a través de mutaciones puntuales.
Ingeniería del andamio de la alfabeta/beta alfa metalohidrolasa de la glyoxalasa II.

Principales resultados:

Se creó una nueva enzima, evMBL8 (beta-lactamasa metálico evolucionado 8), con actividad de la beta-lactamasa.
La enzima modificada perdió su actividad original de glyoxalasa II.
EvMBL8 catalizó de manera eficiente la hidrólisis de la cefotaxima con un (kcat/Km) app de 1,8 x 10 2 (mol/litro) 1 segundo 1.

Conclusiones:

La estrategia de modificar los bucles del sitio activo es efectiva para crear enzimas con nuevas funciones.
El evMBL8 modificado demuestra una actividad significativa de la beta-lactamasa, lo que confiere una resistencia sustancial al crecimiento de Escherichia coli en cefotaxime.

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