Isolation and characterization of a D-cysteine desulfhydrase protein from Arabidopsis thaliana

In several organisms D-cysteine desulfhydrase (D-Mes) activity (EC 4.1.99.4) was measured; this enzyme decomposes D-cysteine into pyruvate, H2S, and NH3. A gene encoding a putative D-Mes protein was identified in Arabidopsis thaliana (L) Heynh. based on high homology to an Escherichia coli protein called YedO that has D-Mes activity. The deduced Arabidopsis protein consists of 401 amino acids and has a molecular mass of 43.9 kDa. It contains a pyridoxal-5'-phosphate binding site. The purified recombinant mature protein had a K,, for D-cysteine of 0.25 mm. Only D-cysteine but not L-cysteine was converted by D-Mes to pyruvate, H2S, and NH3. The activity was inhibited by aminooxy acetic acid and hydroxylamine, inhibitors specific for pyridoxal-5'-phosphate dependent proteins, at low micromolar concentrations. The protein did not exhibit 1-aminocyclopropane-1-carboxylate deaminase activity (EC 3.5.99.7) as homologous bacterial proteins. Western blot analysis of isolated organelles and localization studies using fusion constructs with the green fluorescent protein indicated an intracellular localization of the nuclear encoded D-Mes protein in the mitochondria. D-Mes RNA levels increased with proceeding development of Arabidopsis but decreased in senescent plants; D-Mes protein levels remained almost unchanged in the same plants whereas Specific D-Mes activity was highest in senescent plants. In plants grown in a 12-h light/12-h dark rhythm D-CDes RNA levels were highest in the dark, whereas protein levels and enzyme activity were lower in the dark period than in the light indicating post-translational regulation. Plants grown under low sulfate concentration showed an accumulation Of D-Mes RNA and increased protein levels, the D-CDes activity was almost unchanged. Putative in vivo functions of the Arabidopsis D-Mes protein are discussed.