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

Abstract

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.

An Insight into the Mechanism of Human Cysteine Dioxygenase

“…Our inductively coupled plasma mass spectroscopy analysis of purified recombinant wild-type human CDO yielded an iron incorporation of ϳ68%, which is much higher than 10% or 25% reported by other groups (18,19,31,35). Besides iron, other metal elements were detected in purified recombinant CDO, including zinc (18.1%) and trace amounts of magnesium (0.41%) and manganese (0.25%) (Fig.…”

“…4C). We determined a K m of 3.1 Ϯ 0.3 mM with a k cat of 1.7 s Ϫ1 that is remarkably higher than the k cat for mouse CDO (0.06 s Ϫ1 ) or rat CDO (0.72 s Ϫ1 ) reported previously (18,31,35).…”

“…Oxygen mixed in culture by vigorous shaking is beneficial for cell culture but harmful for maintaining the ferrous state of iron. Because ferric ions cannot be coordinated by the CDO active site, we added exogenous iron just before lysis instead of induction as reported in previous studies (18,31). The harvested pellets were resuspended with 1 mM FeSO 4 and immediately lysed by sonication to release glutathione S-transferase (GST)-fused CDO and mutants, which were purified following standard GST fusion protein purification protocols.…”

The Cysteine Dioxygenase Homologue from Pseudomonas aeruginosa Is a 3-Mercaptopropionate Dioxygenase

“…We previously showed that DL-homocysteine binds to the active site of rat CDO (57). However, the reported extent of inhibition of mammalian CDOs by homocysteine remains ambiguous (17,19,25), and very inefficient use of homocysteine as a substrate has recently been reported (80). Comparable inhibition by cysteamine has been reported for other bacterial CDOs (21).…”

Discovery and Characterization of a Second Mammalian Thiol Dioxygenase, Cysteamine Dioxygenase

“…CDO, for instance, requires ferrous iron for activity (6 -8). Moreover, because of issues with very low iron occupancy (Ͻ10 -25%) after aerobic purification of the enzyme, ferrous iron must be added to assays of CDO to achieve full activity (7,13). Because ADO is also a cupin protein, we wanted to test whether it binds a transition metal and, if so, whether it is required for catalytic activity.…”