RESUMEN
Threonylcarbamoyladenosine (t(6)A) is a universal modification found at position 37 of ANN decoding tRNAs, which imparts a unique structure to the anticodon loop enhancing its binding to ribosomes in vitro. Using a combination of bioinformatic, genetic, structural and biochemical approaches, the universal protein family YrdC/Sua5 (COG0009) was shown to be involved in the biosynthesis of this hypermodified base. Contradictory reports on the essentiality of both the yrdC wild-type gene of Escherichia coli and the SUA5 wild-type gene of Saccharomyces cerevisiae led us to reconstruct null alleles for both genes and prove that yrdC is essential in E. coli, whereas SUA5 is dispensable in yeast but results in severe growth phenotypes. Structural and biochemical analyses revealed that the E. coli YrdC protein binds ATP and preferentially binds RNA(Thr) lacking only the t(6)A modification. This work lays the foundation for elucidating the function of a protein family found in every sequenced genome to date and understanding the role of t(6)A in vivo.
Asunto(s)
Adenosina/análogos & derivados , Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli/genética , ARN de Transferencia/química , Proteínas de Unión al ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Adenosina/biosíntesis , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Genes Esenciales , Genómica , Datos de Secuencia Molecular , ARN de Transferencia/metabolismo , ARN de Transferencia de Treonina/química , ARN de Transferencia de Treonina/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismoRESUMEN
[reaction: see text]. Feeding experiments using a deuterium labeled C16 maleic anhydride and whole cell culture of ATCC 74256 led to the isolation of phomoidride B with deuterium incorporation at C(7) and C(19) as determined by 2H NMR and electrospray mass spectrometry. This result is in accord with a decarboxylative homodimerization of the C16 maleic anhydride as a key biosynthetic step leading to phomoidride B.
Asunto(s)
Anhídridos Maleicos/química , Descarboxilación , Deuterio , Espectroscopía de Resonancia Magnética , Penicillium/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
[reaction: see text]. An intramolecular cyclization strategy for effecting a biomimetic synthesis of the core structure of the fungal secondary metabolites phomoidrides A and B is described. The cyclization substrate 20 is prepared in eight steps from dibromide 10. Treatment of 20 with triethylamine in acetonitrile results in a rapid cyclization to give 21 and 22 in good yield.