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Functional Complementation Analysis (FCA): A Laboratory Exercise Designed and Implemented to Supplement the Teaching of Biochemical Pathways
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El antagonismo conformacional entre sitios activos opuestos en un homólogo bifuncional RelA/SpoT modula el
Tanis Hogg1, Undine Mechold, Horst Malke
1Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Cell
|April 7, 2004
Resumen
Las bacterias sobreviven a la escasez de nutrientes utilizando la respuesta estricta, regulada por las enzimas Rel/Spo. El análisis estructural revela cómo estas enzimas cambian entre sintetizar e hidrolizar la alarmona (p) pGpp.
Área de la Ciencia:
- La bioquímica es la bioquímica.
- Biología Molecular Biología Molecular
- Biología Estructural Biología estructural.
Sus antecedentes:
- Las enzimas Rel/Spo producen la hormona alarmante (p) ppGpp, lo que desencadena la respuesta estricta de las bacterias a la limitación de nutrientes.
- Esta respuesta es crucial para la supervivencia bacteriana durante la inanición.
- Las proteínas Rel/Spo poseen actividad catalítica bifuncional, sintetizando e hidrolizando (p) ppGpp.
Objetivo del estudio:
- Para aclarar la base estructural de la regulación recíproca de las actividades de las enzimas Rel/Spo.
- Para entender el mecanismo de la respuesta estricta a nivel molecular.
Principales métodos:
- Cristalografía de rayos X del homólogo Rel/Spo Rel(Seq) de las subespecies de Streptococcus dysgalactiae. Equisimilis. equisimilis. también conocido como equisimilis.
- Análisis de las conformaciones enzimáticas y estructuras del sitio activo.
- Mutagénesis dirigida al sitio para investigar la regulación enzimática.
Principales resultados:
- La estructura cristalina de 2.1 Å reveló dos conformaciones enzimáticas distintas que corresponden a los estados sintetasa-ON/hidrolasa-OFF y sintetasa-OFF/hidrolasa-ON.
- Los dominios de la hidrolasa y la sintetasa comparten similitudes con las superfamilias de la fosfodiesterasa cíclica y la nucleotidiltransferasa.
- Los sitios activos están distantes pero vinculados, con nucleótidos unidos, incluido el monofosfato cíclico GDP-2':3'-.
- Mutagenesis apoyó la regulación recíproca que involucra la transmisión de señales inducida por ligandos.
Conclusiones:
- La estructura proporciona una base molecular para la regulación recíproca de las actividades de las enzimas Rel/Spo.
- La transmisión de señales inducida por ligandos entre sitios activos probablemente controla la respuesta estricta.
- La comprensión de este mecanismo puede informar estrategias dirigidas a los mecanismos de supervivencia bacteriana.
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