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Characterization of Recombinant Xenopus laevis Type I Iodothyronine Deiodinase: Substitution of a Proline Residue in the Catalytic Center by Serine (Pro132Ser) Restores Sensitivity to 6-Propyl-2-Thiouracil

Department of Internal Medicine (G.G.J.M.K., W.K., T.J.V.), Erasmus Medical Center, 3015 GE Rotterdam, The Netherlands; Laboratory of Comparative Endocrinology (S.V.d.G., V.M.D.), Catholic University Leuven, B3000 Leuven, Belgium; Hubrecht Laboratory (O.D.), Netherlands Institute for Developmental Biology, 3584 CT Utrecht, The Netherlands; and Department Regulations, Development, and Molecular Diversity (G.M.D., B.D.), Museum National d’Histoire Naturelle, Cedex 05, F75231 Paris, France

Address all correspondence and requests for reprints to: Theo J. Visser, Ph.D., Department of Internal Medicine, Room Ee 502, Erasmus Medical Center, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail: t.j.visser{at}erasmusmc.nl.

In frogs such as Rana and Xenopus, metamorphosis does not occur in the absence of a functional thyroid gland. Previous studies indicated that coordinated development in frogs requires tissue and stage-dependent type II and type III iodothyronine deiodinase expression patterns to obtain requisite levels of intracellular T₃ in tissues at the appropriate stages of metamorphosis. No type I iodothyronine deiodinase (D1), defined as T₄ or reverse T₃ (rT3) outer-ring deiodinase (ORD) activity with Michaelis constant (K_m) values in the micromolar range and sensitivity to 6-propyl-2-thiouracil (6-PTU), could be detected in tadpoles so far. We obtained a X. laevis D1 cDNA clone from brain tissue. The complete sequence of this clone (1.1 kb, including poly A tail) encodes an ORF of 252 amino acid residues with high homology to other vertebrate D1 enzymes. The core catalytic center includes a UGA-encoded selenocysteine residue, and the 3' untranslated region (about 300 nt) contains a selenocysteine insertion sequence element. Transfection of cells with an expression vector containing the full-length cDNA resulted in generation of significant deiodinase activity in the homogenates. The enzyme displayed ORD activity with T₄ (K_m 0.5 µM) and rT3 (K_m 0.5 µM) and inner-ring deiodinase activity with T₄ (K_m 0.4 µM). Recombinant Xenopus D1 was essentially insensitive to inhibition by 6-PTU (IC₅₀ > 1 mM) but was sensitive to gold thioglucose (IC₅₀ 0.1 µM) and iodoacetate (IC₅₀ 10 µM). Because the residue 2 positions downstream from the selenocysteine is Pro in Xenopus D1 but Ser in all cloned PTU-sensitive D1 enzymes, we prepared the Pro132Ser mutant of Xenopus D1. The mutant enzyme showed strongly increased ORD activity with T₄ and rT3 (K_m about 4 µM) and was highly sensitive to 6-PTU (IC₅₀ 2 µM). Little native D1 activity could be detected in Xenopus liver, kidney, brain, and gut, but significant D1 mRNA expression was observed in juvenile brain and adult liver and kidney. These results indicate the existence of a 6-PTU-insensitive D1 enzyme in X. laevis tissues, but its role during tadpole metamorphosis remains to be defined.

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				G. Morvan Dubois, A. Sebillot, G. G. J. M. Kuiper, C. H. J. Verhoelst, V. M. Darras, T. J. Visser, and B. A. Demeneix Deiodinase Activity Is Present in Xenopus laevis during Early Embryogenesis Endocrinology, October 1, 2006; 147(10): 4941 - 4949. [Abstract] [Full Text] [PDF]