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Tyrosine, Cysteine, and S-Adenosyl Methionine Stimulate In Vitro [FeFe] Hydrogenase Activation

BACKGROUND: [FeFe] hydrogenases are metalloenzymes involved in the anaerobic metabolism of H(2). These proteins are distinguished by an active site cofactor known as the H-cluster. This unique [6Fe–6S] complex contains multiple non-protein moieties and requires several maturation enzymes for its ass...

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Detalles Bibliográficos
Autores principales: Kuchenreuther, Jon M., Stapleton, James A., Swartz, James R.
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2762031/
https://www.ncbi.nlm.nih.gov/pubmed/19855833
http://dx.doi.org/10.1371/journal.pone.0007565
Descripción
Sumario:BACKGROUND: [FeFe] hydrogenases are metalloenzymes involved in the anaerobic metabolism of H(2). These proteins are distinguished by an active site cofactor known as the H-cluster. This unique [6Fe–6S] complex contains multiple non-protein moieties and requires several maturation enzymes for its assembly. The pathways and biochemical precursors for H-cluster biosynthesis have yet to be elucidated. PRINCIPAL FINDINGS: We report an in vitro maturation system in which, for the first time, chemical additives enhance [FeFe] hydrogenase activation, thus signifying in situ H-cluster biosynthesis. The maturation system is comprised of purified hydrogenase apoprotein; a dialyzed Escherichia coli cell lysate containing heterologous HydE, HydF, and HydG maturases; and exogenous small molecules. Following anaerobic incubation of the Chlamydomonas reinhardtii HydA1 apohydrogenase with S-adenosyl methionine (SAM), cysteine, tyrosine, iron, sulfide, and the non-purified maturases, hydrogenase activity increased 5-fold relative to incubations without the exogenous substrates. No conditions were identified in which addition of guanosine triphosphate (GTP) improved hydrogenase maturation. SIGNIFICANCE: The in vitro system allows for direct investigation of [FeFe] hydrogenase activation. This work also provides a foundation for studying the biosynthetic mechanisms of H-cluster biosynthesis using solely purified enzymes and chemical additives.