CHARACTERIZATION OF A PROPYLTHIOURACIL-INSENSITIVE TYPE-I IODOTHYRONINE DEIODINASE

Mammalian type I iodothyronine deiodinase (D1) activates and inactivat es thyroid hormone by outer ring deiodination (ORD) and inner ring dei odination (IRD), respectively, and is potently inhibited by propylthio uracil (PTU). Here we describe the cloning and characterization of a c omplementary DNA encoding a PTU-insensitive D1 from teleost fish (Oreo chromis niloticus, tilapia). This complementary DNA codes for a protei n of 248 amino acids, including a putative selenocysteine (Sec) residu e, encoded by a TGA triplet, at position 126. The 3' untranslated regi on contains two putative Sec insertion sequence (SECIS) elements. Reco mbinant enzyme expressed in COS-1 cells catalyzes both ORD of T-4 and rT(3) and IRD of T-3 and T-3 sulfate with the same substrate specifici ty as native tilapia D1 (tD1), i.e. rT(3) much greater than T-4 > T-3 sulfate > T-3. Native and recombinant tD1 show equally low sensitiviti es to inhibition by PTU, iodoacetate, and gold thioglucose compared wi th the potent inhibitions observed with mammalian D1s. Because the res idue 2 positions downstream from Sec is Pro in tD1 and in all (PTU-ins ensitive) type II and type III iodothyronine deiodinases but Ser in al l PTU-sensitive D1s, we prepared the Pro128Ser mutant of tD1. The muta nt enzyme showed strongly decreased ORD and somewhat increased IRD act ivity, but was still insensitive to PTU. These results provide new inf ormation about the structure-activity relationship of D1 concerning tw o characteristic properties, i.e. catalysis of both ORD and IRD, and i nhibition by PTU.