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Conversión de una racemasa dependiente de PLP en una aldoasa por una sola mutación en el sitio activo
Florian P Seebeck1, Donald Hilvert
1Laboratorium für Organische Chemie, Swiss Federal Institute of Technology, ETH Hönggerberg, CH-8093 Zürich, Switzerland.
Journal of the American Chemical Society
|August 21, 2003
Resumen
La mutación de la alanina racemasa (Alr) la convierte en una aldolasa eficiente. La enzima mutante Y265A divide selectivamente la beta-fenilserina, ofreciendo información sobre la evolución de la enzima y la síntesis de la pared celular bacteriana.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Enzimología Enzimología.
- Biología Estructural Biología estructural.
Sus antecedentes:
- La alanina racemasa (Alr) es una enzima dependiente de piridoxal 5'-fosfato crucial para la biosíntesis de la pared celular bacteriana.
- Alr cataliza la inversión estereoquímica de la alanina, un paso vital en la formación de peptidoglicanos.
Objetivo del estudio:
- Para investigar las consecuencias funcionales de la mutación de la tirosina 265 (Tyr265) en Geobacillus stearothermophilus alanine racemase.
- Caracterizar la actividad enzimática y la especificidad del sustrato del mutante Y265A, explorando su potencial actividad de aldolasa.
Principales métodos:
- Se empleó mutagénesis dirigida al sitio para generar el mutante Y265A de la alanina racemasa.
- Se realizaron ensayos de cinética enzimática y estereoselectividad para analizar la eficiencia catalítica y la preferencia de sustrato de las enzimas de tipo silvestre y mutantes.
- Se realizó un análisis comparativo de las actividades enzimáticas de tipo silvestre y mutante.
Principales resultados:
- La mutación de Y265A mejoró dramáticamente la eficiencia catalítica de la enzima para la escisión de la beta-fenilserina en 2,3 x 105 veces, convirtiendo a Alr en una aldolasa eficaz.
- La actividad de la racemasa se redujo significativamente en el mutante Y265A.
- La enzima mutante exhibió una alta estereoselectividad para la (2R,3S) -fenilserina, procesando sólo el enantiómero D-aminoácido.
Conclusiones:
- La mutación Y265A demuestra que la alanina racemasa puede convertirse fácilmente en una aldolasa estereoselectiva.
- Se propone que la histidina 166 (His166) funcione como la base catalítica en la reacción retroaldol del mutante.
- Estos hallazgos apoyan un origen evolutivo común para las racemasas de alanina y las aldolasas de D-treonina.
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