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Involvement of the Cys-Tyr cofactor on iron binding in the active site of human cysteine dioxygenase

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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.

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Abbreviations

CDO:

Cysteine dioxygenase

Cys-Tyr cofactor:

Cysteine-tyrosine cofactor

CSA:

Cysteine sulfinic acid

GA:

Glutarate

α-KG:

α-Ketoglutarate

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Acknowledgments

Technical assistance by Simona Jansen, Joana Stegemann and Monika Laurien (University of Cologne, Germany) is gratefully acknowledged. This work was supported by the Friedrich-Ebert-Foundation (to SA) and Center for Molecular Medicine Cologne (CMMC grant D05).

Conflict of interest

The authors do not declare a conflict of interest.

Author information

Authors and Affiliations

  1. Department of Chemistry, Institute of Biochemistry, University of Cologne, 50674, Cologne, Germany

    Sita Arjune, Guenter Schwarz & Abdel A. Belaidi

  2. Center for Molecular Medicine Cologne (CMMC), University of Cologne, 50674, Cologne, Germany

    Sita Arjune, Guenter Schwarz & Abdel A. Belaidi

  3. Cologne Excellence Cluster on Cellular Stress Responses in Ageing-Associated Diseases (CECAD), University of Cologne, 50674, Cologne, Germany

    Guenter Schwarz

  4. Zuelpicher Str. 47, 50674, Cologne, Germany

    Abdel A. Belaidi

Authors
  1. Sita Arjune
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  2. Guenter Schwarz
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  3. Abdel A. Belaidi
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Correspondence to Abdel A. Belaidi.

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Arjune, S., Schwarz, G. & Belaidi, A.A. Involvement of the Cys-Tyr cofactor on iron binding in the active site of human cysteine dioxygenase. Amino Acids 47, 55–63 (2015). https://doi.org/10.1007/s00726-014-1843-7

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  • DOI: https://doi.org/10.1007/s00726-014-1843-7

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