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Peroxiredoxins (Prxs) play important roles in antioxidant defense and redox signaling pathways. A Prx isozyme cDNA (TcPrx2, 745 bp, EF552425) was cloned from Taiwanofungus camphorata and its recombinant protein was overexpressed. The purified protein was shown to exist predominantly as a dimer by sodium dodecyl sulfate-polyacrylamide gel electrolysis in the absence of a reducing agent. The protein in its dimeric form showed no detectable Prx activity. However, the protein showed increased Prx activity with increasing dithiothreitol concentration which correlates with dissociation of the dimer into monomer. The TcPrx2 contains two Cys residues. The Cys located in the conserved active site is the putative active peroxidatic Cys. The role of Cys was investigated by site-directed mutagenesis. The C31S mutant (C → S) exists predominantly as a monomer with noticeable Prx activity. The Prx activity of the mutant was higher than that of the corresponding wild-type protein by nearly twofold at 12 μg/mL. The substrate preference of the mutant was HO > cumene peroxide > t-butyl peroxide. The Michaelis constant ( K) value for HO of the mutant was 0.11 mM. The mutant enzyme was active under a broad pH range from 6 to 10. The results suggest a role of Cys in dimerization of the TcPrx2, a role which, at least in part, may be involved in determining the activity of Prx. The C residue does not function as a resolving Cys and therefore the TcPrx2 must follow the reaction mechanism of 1-Cys Prx. This TcPrx2 represents a new isoform of Prx family. [ABSTRACT FROM AUTHOR]

Abstract: Nitroreductases (Nrs) play important roles in redox system via NADPH or NADH as a reductant. A TcNr cDNA encoding a putative Nr was cloned from Taiwanofungus camphorata. A 3-D structural model of the TcNr has been created based on the known structure of BcNr (Bacillus cereus). To characterise the TcNr, the coding region was subcloned into an expression vector and transformed into Escherichia coli. The recombinant His6-tagged TcNr was purified by Ni affinity chromatography. The purified enzyme showed a single band at molecular mass of approximately 25kDa on 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The enzyme exhibited Nr activity via ferricyanide assay. The Michaelis constant (K M) value for ferricyanide was 0.86mM. The enzyme’s half-life of deactivation at 45°C was 12.3min. The enzyme was most active at pH 6. The enzyme’s preferred substrate is 1-chloro-2, 4-dinitrobenzene. [Copyright &y& Elsevier]

Abstract: Peroxiredoxins (Prxs) are a family of antioxidant peroxidases. The functions of Prxs comprises of cell protection against oxidative stress and regulation of cell proliferation. A putative 2-Cys Prx isozyme (Prx1) cDNA was cloned from Taiwanofungus camphorata (commonly known as Niu-chang-chih in Taiwan). The deduced amino acid sequence is conserved amongst the reported Prxs. A 3-D homology structure was created for this Prx1. To characterise the T. camphorata Prx1, the coding region was subcloned into a pAVD10 and transformed into Escherichia coli. The recombinant 6His-tagged Prx1 was expressed and purified by Ni2+-nitrilotriacetic acid sepharose. The purified enzyme showed two forms using a 15% SDS–PAGE. The enzyme retained 60% activity at 60°C for 2.5min. The enzyme was stable under a broad pH range from 5 to 11. The enzyme showed 57% activity after 40min of incubation at 37°C with trypsin. The ability of the enzyme to protect intact supercoiled plasmid DNA from ·OH induced nicking was demonstrated. [Copyright &y& Elsevier]