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J. Biol. Chem., Vol. 280, Issue 11, 9865-9869, March 18, 2005

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Heterologous Expression, Purification, and Characterization of Recombinant Rat Cysteine Dioxygenase^*

Sergio C. Chai, Ann A. Jerkins¶, Jacob J. Banik, Ilia Shalev, Jennifer L. Pinkham||, Peter C. Uden, and Michael J. Maroney**

From the Departments of Chemistry and ^||Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003 and Department of Nutritional Sciences and the Nutritional Sciences Program, University of Arizona, Tucson, Arizona 85721

Cysteine dioxygenase (CDO, EC 1.13.11.20) catalyzes the oxidation of cysteine to cysteine sulfinic acid, which is the first major step in cysteine catabolism in mammalian tissues. Rat liver CDO was cloned and expressed in Escherichia coli as a 26.8-kDa N-terminal fusion protein bearing a polyhistidine tag. Purification by immobilized metal affinity chromatography yielded homogeneous protein, which was catalytically active even in the absence of the secondary protein-A, which has been reported to be essential for activity in partially purified native preparations. As compared with those existing purification protocols for native CDO, the milder conditions used in the isolation of the recombinant CDO allowed a more controlled study of the properties and activity of CDO, clarifying conflicting findings in the literature. Apo-protein was inactive in catalysis and was only activated by iron. Metal analysis of purified recombinant protein indicated that only 10% of the protein contained iron and that the iron was loosely bound to the protein. Kinetic studies showed that the recombinant enzyme displayed a K_m value of 2.5 ± 0.4 mM at pH 7.5 and 37 °C. The enzyme was shown to be specific for L-cysteine oxidation, whereas homocysteine inhibited CDO activity.

Received for publication, December 6, 2004 , and in revised form, December 22, 2004.

^* This work was funded in part by National Institutes of Health Grant R03-DK-50933 and United States Department of Agriculture Grants ARZT-136722-H-23-114 and ARZT-136778-H-23-123. Trainee funding (to S. C. C.) was provided by the National Institutes of Health-Chemistry Biology Interface Program, Grant T32 GM08515. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This article is dedicated to Dr. Jennifer Pinkham, who passed away February 1, 2005.

^¶ Current address: 6701 Rockledge Dr., Rm. 6154 (MSC 7892), Bethesda, MD 20892.

Deceased.

^** To whom correspondence should be addressed: Dept. of Chemistry, Lederle Graduate Research Tower, Rm. 701, University of Massachusetts, 710 N. Pleasant St., Amherst, MA 01003. Tel.: 413-545-4876; Fax: 413-545-4490; E-mail: mmaroney{at}chem.umass.edu.

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