RESUMO
Imidazole glycerol phosphate synthase (HisFH) is a heterodimeric bienzyme complex operating at a central branch point of metabolism. HisFH is responsible for the HisH-catalyzed hydrolysis of glutamine to glutamate and ammonia, which is then used for a cyclase reaction by HisF. The HisFH complex is allosterically regulated but the underlying mechanism is not well understood. Here, we elucidate the molecular basis of the long range, allosteric activation of HisFH. We establish that the catalytically active HisFH conformation is only formed when the substrates of both HisH and HisF are bound. We show that in this conformation an oxyanion hole in the HisH active site is established, which rationalizes the observed 4500-fold allosteric activation compared to the inactive conformation. In solution, the inactive and active conformations are in a dynamic equilibrium and the HisFH turnover rates correlate with the population of the active conformation, which is in accordance with the ensemble model of allostery.
Assuntos
Regulação Alostérica , Aminoidrolases/química , Aminoidrolases/metabolismo , Aminoidrolases/genética , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Glutamina/metabolismo , Hidrólise , Imidazóis/metabolismo , Espectroscopia de Ressonância Magnética , Complexos Multienzimáticos , Mutação , Conformação Proteica , Ribonucleotídeos/metabolismo , Thermotoga maritima/enzimologiaRESUMO
Direct repair of UV-induced DNA lesions represents an elegant method for many organisms to deal with these highly mutagenic and cytotoxic compounds. Although the participating proteins are structurally well investigated, the exact repair mechanism of the photolyase enzymes remains a vivid subject of current research. In this review, we summarize and highlight the recent contributions to this exciting field.
Assuntos
Dano ao DNA/efeitos dos fármacos , Reparo do DNA , Desoxirribodipirimidina Fotoliase/metabolismo , Dímeros de Pirimidina/química , Raios Ultravioleta , Cristalografia por Raios X , Citotoxinas/toxicidade , Estrutura Molecular , Mutagênicos/toxicidadeRESUMO
The spore photoproduct lyase is a radical SAM enzyme, which repairs 5-(α-thyminyl)-5,6-dihydrothymidine. Here we show that the enzyme establishes a complex radical transfer cascade and creates a cysteine and a tyrosyl radical dyade to establish repair. This allows the enzyme to solve topological and energetic problems associated with the radical based repair reaction.
Assuntos
Reparo do DNA , Proteínas/metabolismo , Timidina/análogos & derivados , Cisteína/química , Geobacillus/enzimologia , Modelos Químicos , Esporos Bacterianos/efeitos da radiação , Timidina/química , Tirosina/químicaRESUMO
Useful diversity: Quantification of modified tRNA nucleobases in different murine and porcine tissues reveals a tissue-specific overall modification content. The modification content correlates with rates of protein synthesis inâ vitro, suggesting a direct link between tRNA modification levels and tissue-specific translational efficiency.
Assuntos
Deutério/química , Nucleosídeos/análise , RNA de Transferência/análise , Animais , Isótopos de Carbono , Cromatografia Líquida , Marcação por Isótopo , Espectrometria de Massas , Camundongos , Isótopos de Nitrogênio , Nucleosídeos/metabolismo , RNA de Transferência/metabolismo , Ribonuclease Pancreático/metabolismo , SuínosRESUMO
UV light is one of the major causes of DNA damage. In spore DNA, due to an unusual packing of the genetic material, a special spore photoproduct lesion (SP lesion) is formed, which is repaired by the enzyme spore photoproduct lyase (Spl), a radical S-adenosylmethionine (SAM) enzyme. We report here the synthesis and DNA incorporation of a DNA SP lesion analogue lacking the phosphodiester backbone. The oligonucleotides were used for repair studies and they were cocrystallized with a polymerase enzyme as a template to clarify the configuration of the SP lesion and to provide information about the base-pairing properties of the lesion. The structural analysis together with repair studies allowed us to clarify the identity of the preferentially repaired lesion diastereoisomer.