Iodotyrosine Deiodinase Is the First Mammalian Member of the NADH Oxidase/Flavin Reductase Superfamily

Abstract

The enzyme responsible for iodide salvage in the thyroid, iodotyrosine deiodinase, was solubilized from porcine thyroid microsomes by limited proteolysis with trypsin. The resulting protein retained deiodinase activity and was purified using anion exchange, dye, and hydrophobic chromatography successively. Peptide sequencing of the final isolate identified the gene responsible for the deiodinase. The amino acid sequence of the porcine enzyme is highly homologous to corresponding genes in a variety of mammals including humans, and the mouse gene was expressed in human embryonic kidney 293 cells to confirm its identity. The amino acid sequence of the deiodinase suggests the presence of three domains. The N-terminal domain provides a membrane anchor. The intermediate domain contains the highest sequence variability and lacks homology to structural motifs available in the common databases. The C-terminal domain is highly conserved and resembles bacterial enzymes of the NADH oxidase/flavin reductase superfamily. A three-dimensional model of the deiodinase based on the coordinates of the minor nitroreductase of Escherichia coli indicates that a Cys common to all of the mammal sequences is located adjacent to bound FMN. However, the deiodinase is not structurally related to other known flavoproteins containing redox-active cysteines or the iodothyronine deiodinases containing an active site selenocysteine.

Crystal Structure of Iodotyrosine Deiodinase, a Novel Flavoprotein Responsible for Iodide Salvage in Thyroid Glands

“…In the absence of substrate, IYD crystallized with one monomer per asymmetric unit. However, analysis of the crystal packing interactions revealed an extensive interface with a symmetry-related molecule (2490 Å 2 per monomer) indicating that the enzyme forms a domain-swapped dimer as observed in related proteins within the same structural superfamily (4,21). The IYD⅐MIT complex crystallized with two monomers per asymmetric unit, and the IYD⅐DIT complex crystallized with eight monomers per asymmetric unit.…”

“…Such mutants are particularly tragic because their detection often occurs only after developmental damage to patients has occurred (1). Studies on IYD to date have been limited by its membrane association and transient expression in mammalian cells (3,4). However, deletion of its N-terminal membrane anchor yields a soluble and active enzyme (6) that has been expressed in Sf9 cells in sufficient quantities to support the crystallographic analysis reported here.…”

“…Iodothyronine deiodinase is a member of the thioredoxin structural superfamily and utilizes an active site selenocysteine for its catalysis. In contrast, IYD is a member of the NADH oxidase/flavin reductase superfamily (3,4) and requires neither selenocysteine nor cysteine for catalysis (6). Instead a bound flavin mononucleotide (FMN) is required (7).…”

“…IYD catalyzes a reductive deiodination of MIT and DIT selectively to prevent loss of iodide that would otherwise occur by excretion of these amino acids. The gene encoding IYD has recently been identified (3,4) and has provided an initial basis for correlating its mutation with hypothyroidism and goiter observed in certain patients (1). The crystal structure described in this work now supersedes the previous structural models.…”

In vitro metabolic stability of iodinated obestatin peptides

“…Dehalogenation of MIT by thyroid iodotyrosine deiodinase (IYD) was reported to be faster than that of DIT [23], and requires a bound flavin mononucleotide (FMN) for its catalytic activity. In contrast, iodothyronine deiodinase (ID or DI) requires an active-site Cys for reduced thyroxine generation [7]. These enzymes represent the most common two currently known reductive dehalogenation pathways in mammals (Fig.…”