Structure-Based Insights into the Role of the Cys–Tyr Crosslink and Inhibitor Recognition by Mammalian Cysteine Dioxygenase (original) (raw)

In mammals, the non-heme iron enzyme cysteine dioxygenase (CDO) helps regulate cysteine (Cys) levels through converting Cys to cysteine sulfinic acid (CSA). Its activity is in part modulated by the formation of a Cys93-Tyr157 crosslink that increases its catalytic efficiency over 10-fold. Here, 21 high-resolution mammalian CDO structures are used to gain insight into how the Cys-Tyr crosslink promotes activity and how select competitive inhibitors bind. Crystal structures of crosslink-deficient C93A and Y157F variants reveal similar ~1.0 Å shifts in the side chain of residue 157, and both variant structures have a new chloride ion coordinating the active site iron. Cys binding is also different than for wild-type CDO and no Cys-persulfenate forms in the C93A or Y157F active sites at either pH=6.2 or 8.0. We conclude that the crosslink enhances activity by positioning the Tyr157 hydroxyl for enabling proper Cys binding, proper oxygen binding, and optimal chemistry. In addition, structures are presented for homocysteine, thiosulfate and azide bound as competitive inhibitors. The observed binding modes of homocysteine and D-Cys make clear why they are not substrates, and the binding of azide shows that, in contrast to what has been proposed, it does not bind in these crystals as a superoxide mimic.