Three O -acetyl-L-serine(thiol)lyase isoenzymes from Arabidopsis catalyse cysteine synthesis and cysteine desulfuration at different pH values

So far, 7 different genes of the β-substituted alanine synthase family coding for proteins with O-acetyl-L-serine(thiol)lyase activity (OAS-TL) have been identified in Arabidopsis. The differences with respect to function and regulation of these isoforms are not well understood. In the present work, we aimed to differentiate between three OAS-TL proteins, A, B, and C, which are presumably localized in the cytoplasm, in plastids, and in mitochondria, respectively. The mature proteins were expressed in E. coli and the pH optima for the formation of cysteine were determined to pH 8.0, pH 8.0, and pH 7.0, respectively. The purified recombinant OAS-TL proteins also catalysed the formation of hydrogen sulfide (H2S) and β-cyanoalanine from cysteine in the presence of dithiothreitol or KCN, respectively. The three isoenzymes differed in their Km for cysteine in the presence of dithiothreitol, but the Km for cysteine in the presence of KCN were almost identical. On a molar basis, the formation of H2S or cysteine by the OAS-TL proteins varied in relation to pH. For the determination of both enzyme activities, cysteine synthesis and cysteine desulfuration, in different compartments of plant cells, chloroplasts and mitochondria were isolated from Arabidopsis plants and the OAS-TL and L-cysteine desulfhydrase activities were determined. In conclusion, a) the evolution of H2S from higher plants originates from different enzyme reactions, and b) the side reactions of the OAS-TL proteins seem to contribute massively to the total H2S release of higher plants at least at higher pH values.