Heterologous expression, purification, and characterization of recombinant rat cysteine dioxygenase.
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 degrees C. The enzyme was shown to be specific for l-cysteine oxidation, whereas homocysteine inhibited CDO activity.
Chai
Sergio C
SC
Department of Chemistry and Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Jerkins
Ann A
AA
Banik
Jacob J
JJ
Shalev
Ilia
I
Pinkham
Jennifer L
JL
Uden
Peter C
PC
Maroney
Michael J
MJ
D000818:Animals; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D050558:Cysteine Dioxygenase; D018076:DNA, Complementary / Q000378:metabolism; D049308:Dioxygenases / Q000737:chemistry* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006710:Homocysteine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D016366:Open Reading Frames; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013696:Temperature; D013997:Time Factors
D018076:DNA, Complementary; D011994:Recombinant Proteins; D006710:Homocysteine; D006639:Histidine; D007501:Iron; D049308:Dioxygenases; D050558:Cysteine Dioxygenase; D003545:Cysteine; D010100:Oxygen; C013461:cysteine sulfinic acid