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Background Thioredoxin (Trx) is reduced by thioredoxin reductase. Trx is used in ribonucleoide reduction, assimilatory sulfate reduction, in modulation of protein sulfhydryl groups, and refolding proteins. Results A TcTrx (Tc: Taiwanofungus camphorata) cDNA (640 bp, GenBank AY838902.1) encoding a putative thioredoxin (Trx) of 135 amino acid residues with calculated molecular mass of 16.17 kDa was cloned from Taiwanofungus c amphorata. The deduced amino acid sequence containing a motif (Cys-Gly-Pro-Cys) that is highly conserved among the reported Trxs. A three dimensional structural model of the TcTrx has been created based on the known structure of Malassezia sympodialis Trx (MsTrx, PDB ID: 2j23). To characterize the TcTrx, the codon optimized coding region was subcloned into an expression vector and transformed into Saccharomyces cerevisiae. The recombinant His8-tagged TcTrx was expressed and purified by Ni affinity chromatography. The purified enzyme showed a band of approximately 32 kDa (expected dimeric form) on a 12% SDS-PAGE. The molecular mass determined by MALDI-TOF is 33.16 kDa which suggests that the purified enzyme is a dimeric enzyme. Furthermore, the enzyme exhibited TcTrx activity via insulin assay. The Michaelis constant (K M ) value for insulin was 3.78 × 10−2 mM. The enzyme’s half-life of deactivation was 13 min at 45°C. The enzyme was most active at pH 7. Conclusions A three dimensional structural model of T. camphorata Trx based on its TcTrx cDNA sequence. The active form of the TcTrx has been successfully expressed in yeast. The enzyme possesses Trx activity and is capable of reduction of disulfide bonds during the formation of newly synthesized proteins.

Background Aryl-alcohol dehydrogenases (AADs) have been known to involve in the metabolism of aromatic compounds. Results One TcAAD cDNA (GenBank HQ453361) encoding a putative aryl-alcohol dehydrogenase (AAD) was cloned from Taiwanofungus camphorata. The deduced amino acid sequence is conserved among the reported AADs. A 3-D structural model of the TcAAD has been created based on the known structure of voltage-dependent potassium channels subunit beta-2 (PDB code: 3EAU). To characterize the TcAAD, the coding region was subcloned into an expression vector and transformed into Saccharomyces cerevisiae. The recombinant His6-tagged TcAAD was overexpressed and purified by Ni affinity chromatography. The purified enzyme showed a band of approximately 39 kDa on a 12% SDS-PAGE. The molecular mass determined by MALDI-TOF is 40.58 kDa which suggests that the purified enzyme is a monomeric enzyme. Using veratraldehyde as a substrate, the K M, Vmax of TcADD was determined at pH 6.0. Using benzyl alcohol derivatives as substrates, the oxidizing power of TcADD via NAD+ at pH 9.6 was studied. Conclusions The coding sequence of the TcAAD cDNA was introduced into an S. cerevisiae expression system and the active enzyme purified and characterized. Understanding the properties of this TcAAD will be beneficial for its potential in xenobiotic detoxification or production of natural flavors.