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Biosíntesis de tiamina tiazol en eucariotas: conversión de NAD en un intermediario avanzado
Abhishek Chatterjee1, Christopher T Jurgenson, Frank C Schroeder
1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA.
Journal of the American Chemical Society
|February 21, 2007
Resumen
Los investigadores identificaron mutantes parcialmente activos de la tiazol sintasa (THI4) que utilizan el nicotinamida adenina dinucleótido (NAD) como sustrato. Este hallazgo aclara las primeras etapas en la biosíntesis de la fracción tiazol del pirofosfato de tiamina en eucariotas.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Enzimología Enzimología.
- Biología Molecular Biología Molecular
Sus antecedentes:
- La tiazol sintasa (THI4) de Saccharomyces cerevisiae es crucial para sintetizar la porción tiazol del pirofosfato de tiamina.
- El THI4 de tipo silvestre copurifica con metabolitos adenilados, lo que dificulta los esfuerzos para comprender su mecanismo catalítico.
- Los intentos anteriores de activar el THI4 de tipo silvestre mediante la liberación de metabolitos unidos no tuvieron éxito.
Objetivo del estudio:
- Identificar y caracterizar mutantes activos de THI4 para dilucidar su sustrato y su mecanismo catalítico.
- Para investigar las primeras etapas en la biosíntesis de la fracción tiamina tiazol en eucariotas.
Principales métodos:
- La mutagénesis dirigida al sitio se utilizó para crear mutantes C204A y H200N THI4.
- Se realizaron ensayos de actividad enzimática para detectar la liberación de metabolitos y la formación de intermedios.
- La caracterización de los intermedios de reacción se logró utilizando espectroscopia de RMN y ESI-MS después de atrapar con ortofenilendiamina.
Principales resultados:
- Se identificaron dos mutantes THI4 parcialmente activos (C204A y H200N).
- Estos mutantes liberaron la fracción de nicotinamida del dinucleótido de nicotinamida adenina (NAD), produciendo ADP-ribosa y posteriormente ADP-ribulosa.
- En presencia de glicina, los mutantes formaron un intermediario avanzado, que fue atrapado y caracterizado.
Conclusiones:
- El nicotinamida adenina dinucleótido (NAD) se confirma como un sustrato para THI4.
- El estudio aclara los pasos iniciales de la biosíntesis única de la fracción tiamina tiazol en los organismos eucariotas.
- La caracterización mutante proporciona nuevos conocimientos sobre el mecanismo catalítico de la tiazol sintasa.
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