Glutathione-homocystine transhydrogenase/oxidoreductase (GHTHase) is an enzyme that catalyzes the reversible oxidation/reduction of reduced glutathione (GSH) and homocystine (Hcy2) to oxidized glutathione and homocysteine (Hcy). GHTHase is implicated in regulation of the metabolism of homocysteine and glutathione. Aspergillus flavipes JF831014 exhibits the highest productivity of GHTHase, using GSH as electron donor and Hcy2 as acceptor. GHTHase yield from A. flavipes has been nutritionally optimized to reach the maximum activity (21.14 U/mg) using GSH (0.4%) combined with Hcy2 (0.01%) and glucose (0.4%), NADH + H (30 mM) at medium initial pH 7.8. The yield of GHTHase was increased about 1.2 times upon starvation of the culture of A. flavipes for two days, as compared to the non-sulfur starved culture. The GHTHase activity was increased by 13.7 fold with total yield of 8.8%. According to denaturing PAGE, GHTHase had 35 kDa, and 75 kDa by non-denaturing PAGE, gel-filtration and DLS analysis, ensuring its homodimeric identity. The enzyme displayed a highest activity at pH 6.5 – 7.6, 40°C, and pH stability within 6.0 – 8.0. GHTHase has higher affinity for GSH (K m = 14.3 mM) and cysteine (K m = 15.1 mM) as hydrogen donor for Hcy2 reduction, other than CDNB for glutathione S-transferase. The variant catalytic response to standard glutathione S-transferase substrates, esteem the unique catalytic properties of GHTHase. The enzyme was significantly inhibited by iodoacetate, hydroxylamine, and propargylglycine, revealing their sulfur active site dependence. Thus, a new isoform of glutathione S-transferase, GHTHase, with unique potency to reduce Hcy2 to soluble Hcy using GSH as hydrogen donor, was discovered. The specificity of GHTHase to oxidize toxic insoluble Hcy2 (cardiovascular disorder risk factor) can be a novel route to attack cardiovascular diseases. … Read more