ABSTRACT
Cysteine desulfhydrases decompose cysteine to produce pyruvate, ammonium, and hydrogen sulfide. Using d-cysteine (D-cys) as a substrate, an enzyme with this activity was purified from rice seeds and identified at the native protein level. MALDI-TOF-MS analysis of its tryptic peptides revealed a 426 amino acid protein encoded by the OsDCD1 gene (Os02g0773300). Recombinant OsDCD1 (rOsDCD1) was expressed in Escherichia coli cells and purified as a single protein by column chromatography. Gel filtration column chromatography indicated that the native enzyme was a homodimer. The enzyme exhibited maximum catalytic activity at approximately pH 7.5 and 40 °C and was stable at pH 5.5-7.5 and < 37 °C. Kinetics analysis indicated Km and Vmax values for D-cys of 136 µM and 45.5 µmol/min/mg protein, respectively. In contrast, l-cysteine (L-cys) acted as an inhibitor with mixed non-competitive inhibition. Based on the substrate specificity of rOsDCD1, the amount of D-cys in rice flour was quantified. Even in the presence of up to 1 mM L-cys, the quantification of low concentrations of D-cys was unaffected. We demonstrate for the first time that the amount of D-cys in rice flour varies in the range of 0.76-0.93 µmol/g depending on the variety.
