RESUMO

A novel Cu/ZnSOD from Amaranthus hypochondriacus was cloned, expressed, and characterized. Nucleotide sequence analysis showed an open reading frame (ORF) of 456 bp, which was predicted to encode a 15.6-kDa molecular weight protein with a pI of 5.4. Structural analysis showed highly conserved amino acid residues involved in Cu/Zn binding. Recombinant amaranth superoxide dismutase (rAhSOD) displayed more than 50 % of catalytic activity after incubation at 100 °C for 30 min. In silico analysis of Amaranthus hypochondriacus SOD (AhSOD) amino acid sequence for globularity and disorder suggested that this protein is mainly disordered; this was confirmed by circular dichroism, which showed the lack of secondary structure. Intrinsic fluorescence studies showed that rAhSOD undergoes conformational changes in two steps by the presence of Cu/Zn, which indicates the presence of two binding sites displaying different affinities for metals ions. Our results show that AhSOD could be classified as an intrinsically disordered protein (IDP) that is folded when metals are bound and with high thermal stability.

Assuntos

Amaranthus/enzimologia , Proteínas Intrinsicamente Desordenadas/metabolismo , Superóxido Dismutase/metabolismo , Sequência de Aminoácidos , Cromatografia em Gel , Dicroísmo Circular , Estabilidade Enzimática/efeitos dos fármacos , Fluorescência , Peróxido de Hidrogênio/farmacologia , Proteínas Intrinsicamente Desordenadas/química , Cinética , Metais/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Multimerização Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Cloreto de Sódio/farmacologia , Superóxido Dismutase/química , Temperatura