Cloning and functional characterization of a typical 2-Cys peroxiredoxin from southern bluefin tuna (Thunnus maccoyii).
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 K(m)(Trx) was 12 microM and the S(0.5) values for H2O2 and CuOOH were 29 and 25 microM, 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.
Sutton
Drew L
DL
School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide SA5001, Australia.
Loo
Grace H
GH
Menz
R Ian
RI
Schuller
Kathryn A
KA
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003545:Cysteine / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D054464:Peroxiredoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D016415:Sequence Alignment; D014413:Tuna / Q000235:genetics / Q000378:metabolism*
D029941:Fish Proteins; D054464:Peroxiredoxins; D003545:Cysteine