Animals use three different enzymes to produce de novo hydrogen sulfide, namely cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CGL) and mercaptopyruvate sulfur-transferase (MST). Interestingly, data from whole genome sequencing of the roundworm C. elegans predicted a large number of genes encoding these enzymes (2 genes for CBS, 3 genes for CGL and 7 genes for MST), and moreover, predicted 4 genes encoding O-acetylserine sulfhydrylases. These enzymes serve in plants and bacteria in the sulfur assimilation pathway to produce cysteine from sulfide but some paralogs may detoxify cyanide utilizing cysteine as the substrate to produce beta-cyanoalanine and hydrogen sulfide. To explore whether C. elegans possesses, in contrast to the other animals, an additional class of enzymes producing hydrogen sulfide we aimed at characterizing the roles of O-acetylserine sulfhydrylases, namely CYSL-1 - CYSL-4, especially the catalytic activities of recombinant proteins. We show that despite their shared ancestry nematode proteins are conformationally different and exhibit distinct enzymatic properties indicating that these proteins have diverged in nematode species to adopt distinct functions in vivo. We have determined that one of these proteins - CYSL-2 - can produce hydrogen sulfide when utilizing cysteine and cyanide as the substrates and available data indicate the role of this enzyme in the cyanide detoxification pathway. Since we demonstrated that CYSL-2 can both utilize and produce hydrogen sulfide we hypothesize that this enzyme may also maintain hydrogen sulfide homeostasis in the cells. CYSL-1 has been proven to interact with the proline hydroxylase EGL-9 activating hypoxia-inducible factor HIF-1 as a consequence of the increased levels of hydrogen sulfide in the cells during hypoxia. On the other hand, the role of CYSL-3 remains unclear, however, its biochemical properties suggest that this protein might serve as an enzyme metabolizing S-sulfocysteine and sulfide in these nematodes; expression of
cysl-4 has not been detected. Taken together, our data demonstrate that the roles of O-acetylserine sulfhydrylases in C. elegans are different and that C. elegans, in contrast to mammals, possesses four typologically different enzymes to produce hydrogen sulfide. This study was supported by the research program of the Charles University in Prague (PRVOUK - P24/LF1/3).