Cloning and functional characterization of a typical 2-Cys peroxiredoxin from southern bluefin tuna (Thunnus maccoyii)

Drew Sutton, Hsi-Mei Loo, Robert Menz, Kathryn Schuller

    Research output: Contribution to journalArticle

    21 Citations (Scopus)

    Abstract

    Peroxiredoxins (Prxs, EC: 1.11.1.15) are cysteine-dependent peroxidases proposed to function as antioxidant enzymes and also in H2O2-mediated cell signaling. They have been well characterized in yeast, mammals, protists and bacteria but not yet in fish. Here we describe the cloning and functional characterization of a Prx 2 cDNA from southern bluefin tuna (SBT, Thunnus maccoyii), an important aquaculture species in South Australia. The SBT Prx sequence was closely related (76-92% identical) to Prx 1 and 2 sequences from other fish and mammals and phylogenetic analyses showed that it was most likely a Prx 2. The deduced amino acid sequence contained the peroxidatic and resolving Cys residues characteristic of typical 2-Cys Prx proteins from all kingdoms of life. It also contained the GGLG motif associated with the sensitivity of eukaryotic typical 2-Cys Prx proteins to overoxidation and consequent inactivation by H2O2. When the SBT Prx 2 was expressed in E. coli, it showed thioredoxin (Trx)-dependent peroxidase activity with H2O2, cumene hydroperoxide (CuOOH) and t-butyl hydroperoxide (t-bOOH). The SBT Prx displayed Michaelis-Menten kinetics with Trx but sigmoidal kinetics with H2O2 and CuOOH. The Km(Trx) was 12μM and the S0.5 values for H2O2 and CuOOH were 29 and 25μM, respectively. At mM concentrations of H2O2, SBT Prx progressively lost its peroxidase activity as has been observed for other eukaryotic typical 2-Cys Prx proteins. The native SBT Prx enzyme existed as a mixture of dimers, tetramers, decamers and a higher order aggregate.

    Original languageEnglish
    Pages (from-to)97-106
    Number of pages10
    JournalComparative Biochemistry and Physiology B-Biochemistry and Molecular Biology
    Volume156
    Issue number2
    DOIs
    Publication statusPublished - Jun 2010

    Keywords

    • 2-Cys peroxiredoxin
    • Antioxidant
    • Natural killer enhancing factor
    • NKEF
    • Southern bluefin tuna
    • Thunnus maccoyii

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