RESUMO
Cysteine dioxygenase (CDO, EC 1.13.11.20) catalyzes the oxidation of cysteine to cysteine sulfinic acid, which is the first major step in cysteine catabolism in mammalian tissues. Rat liver CDO was cloned and expressed in Escherichia coli as a 26.8-kDa N-terminal fusion protein bearing a polyhistidine tag. Purification by immobilized metal affinity chromatography yielded homogeneous protein, which was catalytically active even in the absence of the secondary protein-A, which has been reported to be essential for activity in partially purified native preparations. As compared with those existing purification protocols for native CDO, the milder conditions used in the isolation of the recombinant CDO allowed a more controlled study of the properties and activity of CDO, clarifying conflicting findings in the literature. Apo-protein was inactive in catalysis and was only activated by iron. Metal analysis of purified recombinant protein indicated that only 10% of the protein contained iron and that the iron was loosely bound to the protein. Kinetic studies showed that the recombinant enzyme displayed a K(m) value of 2.5 +/- 0.4 mm at pH 7.5 and 37 degrees C. The enzyme was shown to be specific for l-cysteine oxidation, whereas homocysteine inhibited CDO activity.
Assuntos
Cisteína/análogos & derivados , Dioxigenases/química , Dioxigenases/isolamento & purificação , Proteínas Recombinantes/química , Animais , Catálise , Cromatografia Líquida de Alta Pressão , Cisteína/química , Cisteína Dioxigenase , DNA Complementar/metabolismo , Escherichia coli/metabolismo , Histidina/química , Homocisteína/química , Concentração de Íons de Hidrogênio , Ferro/química , Cinética , Modelos Químicos , Fases de Leitura Aberta , Oxigênio/química , Oxigênio/metabolismo , Estrutura Terciária de Proteína , Ratos , Espectrometria de Massas por Ionização por Electrospray , Temperatura , Fatores de TempoRESUMO
Nickel-dependent superoxide dismutases (NiSODs) represent a novel solution to controlling the deleterious effects of reactive oxygen species derived from superoxide in biology. The expression of recombinant Streptomyces coelicolor NiSOD and its in vitro processing and reconstitution to yield fully active enzyme is reported. The results of studies of NiSODs involving mutations in two putative nickel binding ligands are also reported. These studies show that mutation of M28, a strictly conserved residue and one of only three S-donor ligands in the enzyme, has no measurable effect on the spectroscopic or catalytic properties of the enzyme. In contrast, mutation of the strictly conserved N-terminal H residue has dramatic effects on both the spectroscopic and catalytic properties. These results provide insights into structural and mechanistic aspects of the novel nickel-containing reactive site.
