Expression, purification, and kinetic characterization of recombinant rat cysteine dioxygenase, a non-heme metalloenzyme necessary for regulation of cellular cysteine levels.
Cysteine dioxygenase (CDO, EC 1.13.11.20) is a non-heme mononuclear iron enzyme that oxidizes cysteine to cysteinesulfinate. CDO catalyzes the first step in the pathway of taurine synthesis from cysteine as well as the first step in the catabolism of cysteine to pyruvate and sulfate. Previous attempts to purify CDO have been associated with partial or total inactivation of CDO. In an effort to obtain highly purified and active CDO, recombinant rat CDO was heterologously expressed and purified, and its activity profile was characterized. The protein was expressed as a fusion protein bearing a polyhistidine tag to facilitate purification, a thioredoxin tag to improve solubility, and a factor Xa cleavage site to permit removal of the entire N-terminus, leaving only the 200 amino acids inherent to the native protein. A multi-step purification scheme was used to achieve >95% purity of CDO. The approximately 40.3 kDa full-length fusion protein was purified to homogeneity using a three-column scheme, the fusion tag was then removed by digestion with factor Xa, and a final column step was used to purify homogeneous approximately 23 kDa CDO. The purified CDO had high specific activity and kinetic parameters that were similar to those for non-purified rat liver homogenate, including a Vmax of approximately 1880 nmol min-1 mg-1 CDO (kcat=43 min-1) and a Km of 0.45 mM for L-cysteine. The expression and purification of CDO in a stable, highly active form has yielded significant insight into the kinetic properties of this unique thiol dioxygenase.
Simmons
Chad R
CR
Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853-6301, USA.
Hirschberger
Lawrence L
LL
Machi
Mari S
MS
Stipanuk
Martha H
MH
D000818:Animals; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D050558:Cysteine Dioxygenase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008667:Metalloproteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*
D008667:Metalloproteins; D011993:Recombinant Fusion Proteins; D050558:Cysteine Dioxygenase; D003545:Cysteine