title: Involvement of the Cys-Tyr cofactor on iron binding in the active site of human cysteine dioxygenase.
abstract: Sulfur metabolism has gained increasing medical interest over the last years. In particular, cysteine dioxygenase (CDO) has been recognized as a potential marker in oncology due to its altered gene expression in various cancer types. Human CDO is a non-heme iron-dependent enzyme, which catalyzes the irreversible oxidation of cysteine to cysteine sulfinic acid, which is further metabolized to taurine or pyruvate and sulfate. Several studies have reported a unique post-translational modification of human CDO consisting of a cross-link between cysteine 93 and tyrosine 157 (Cys-Tyr), which increases catalytic efficiency in a substrate-dependent manner. However, the reaction mechanism by which the Cys-Tyr cofactor increases catalytic efficiency remains unclear. In this study, steady-state kinetics were determined for wild type CDO and two different variants being either impaired or saturated with the Cys-Tyr cofactor. Cofactor formation in CDO resulted in an approximately fivefold increase in k cat and tenfold increase in k cat/K m over the cofactor-free CDO variant. Furthermore, iron titration experiments revealed an 18-fold decrease in K d of iron upon cross-link formation. This finding suggests a structural role of the Cys-Tyr cofactor in coordinating the ferrous iron in the active site of CDO in accordance with the previously postulated reaction mechanism of human CDO. Finally, we identified product-based inhibition and α-ketoglutarate and glutarate as CDO inhibitors using a simplified well plate-based activity assay. This assay can be used for high-throughput identification of additional inhibitors, which may contribute to understand the functional importance of CDO in sulfur amino acid metabolism and related diseases.
authors.affiliation: Department of Chemistry, Institute of Biochemistry, University of Cologne, 50674, Cologne, Germany.
authors.forename: Sita
authors.initials: S
authors.lastname: Arjune
authors.forename: Guenter
authors.initials: G
authors.lastname: Schwarz
authors.forename: Abdel A
authors.initials: AA
authors.lastname: Belaidi
mesh_terms: D020134:Catalytic Domain; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism*; D050558:Cysteine Dioxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007501:Iron / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D011485:Protein Binding
chemical_list: D003067:Coenzymes; D004151:Dipeptides; C072331:cysteinyltyrosine; D007501:Iron; D050558:Cysteine Dioxygenase