Thiol dioxygenases are non-heme mononuclear-iron proteins and belong to the cupin superfamily. In 2014, mercaptosuccinate dioxygenase (Msdo) of Variovorax paradoxus B4 was identified as another bacterial cysteine dioxygenase (Cdo) homolog catalyzing the conversion of mercaptosuccinate (MS) into succinate and sulfite. To gain further insights into potentially important amino acid residues for enzyme activity, seven enzyme variants were generated and analyzed. (i) Three variants comprised the substitution of one conserved histidine residue each by leucine, either supposed to be mandatory for coordination of the Fe(II) cofactor (H93 and H95) or to be important for substrate positioning within the active site (H163). The corresponding enzyme variants were completely inactive confirming their essential roles for enzyme activity. (ii) Mutation C100S resulted as well in an inactive enzyme demonstrating its importance for either stability or activity of the protein. (iii) For eukaryotic Cdo, a hydrogen bond network for substrate positioning was postulated, and the corresponding amino acids are basically present in Msdo. Albeit the MsdoQ64A mutation exhibited an increased K-m of 0.29 mM when compared to the wildtype with 0.06 mM, it did not significantly affect the specific activity. (iv) The variant MsdoR66A showed only very low activity even when high amounts of enzyme were applied indicating that this residue might be important for catalysis. (v) No strong effect had the mutation Y165F for which a specific enzyme activity of 10.22 mu mol min(-1) mg(-1) protein and a K-m value of 0.06 mM with high similarity to those of the wildtype enzyme were obtained. This residue corresponds to Y157 of human Cdo, which is part of the catalytic triad and is supposed to be involved in substrate positioning. Apparently, another residue could fulfill this role in Msdo, since the loss of Y165 did not have a strong effect.