Publications

• A switch between one- and two-electron chemistry of the human flavoprotein iodotyrosine deiodinase is controlled by substrate.

  Hu J., Chuenchor W., Rokita S.E.

  Reductive dehalogenation is not typical of aerobic organisms but plays a significant role in iodide homeostasis and thyroid activity. The flavoprotein iodotyrosine deiodinase (IYD) is responsible for iodide salvage by reductive deiodination of the iodotyrosine derivatives formed as byproducts of t ... >> More

  Reductive dehalogenation is not typical of aerobic organisms but plays a significant role in iodide homeostasis and thyroid activity. The flavoprotein iodotyrosine deiodinase (IYD) is responsible for iodide salvage by reductive deiodination of the iodotyrosine derivatives formed as byproducts of thyroid hormone biosynthesis. Heterologous expression of the human enzyme lacking its N-terminal membrane anchor has allowed for physical and biochemical studies to identify the role of substrate in controlling the active site geometry and flavin chemistry. Crystal structures of human IYD and its complex with 3-iodo-l-tyrosine illustrate the ability of the substrate to provide multiple interactions with the isoalloxazine system of FMN that are usually provided by protein side chains. Ligand binding acts to template the active site geometry and significantly stabilize the one-electron-reduced semiquinone form of FMN. The neutral form of this semiquinone is observed during reductive titration of IYD in the presence of the substrate analog 3-fluoro-l-tyrosine. In the absence of an active site ligand, only the oxidized and two-electron-reduced forms of FMN are detected. The pH dependence of IYD binding and turnover also supports the importance of direct coordination between substrate and FMN for productive catalysis. << Less

  J. Biol. Chem. 290:590-600(2015) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Functional analysis of iodotyrosine deiodinase from drosophila melanogaster.

  Phatarphekar A., Rokita S.E.

  The flavoprotein iodotyrosine deiodinase (IYD) was first discovered in mammals through its ability to salvage iodide from mono- and diiodotyrosine, the by-products of thyroid hormone synthesis. Genomic information indicates that invertebrates contain homologous enzymes although their iodide requir ... >> More

  The flavoprotein iodotyrosine deiodinase (IYD) was first discovered in mammals through its ability to salvage iodide from mono- and diiodotyrosine, the by-products of thyroid hormone synthesis. Genomic information indicates that invertebrates contain homologous enzymes although their iodide requirements are unknown. The catalytic domain of IYD from Drosophila melanogaster has now been cloned, expressed and characterized to determine the scope of its potential catalytic function as a model for organisms that are not associated with thyroid hormone production. Little discrimination between iodo-, bromo-, and chlorotyrosine was detected. Their affinity for IYD ranges from 0.46 to 0.62 μM (K_d ) and their efficiency of dehalogenation ranges from 2.4 - 9 x 10³ M^-1 s^-1 (k_cat /K_m ). These values fall within the variations described for IYDs from other organisms for which a physiological function has been confirmed. The relative contribution of three active site residues that coordinate to the amino acid substrates was subsequently determined by mutagenesis of IYD from Drosophila to refine future annotations of genomic and meta-genomic data for dehalogenation of halotyrosines. Substitution of the active site glutamate to glutamine was most detrimental to catalysis. Alternative substitution of an active site lysine to glutamine affected substrate affinity to the greatest extent but only moderately affected catalytic turnover. Substitution of phenylalanine for an active site tyrosine was least perturbing for binding and catalysis. << Less

  Protein Sci. 25:2187-2195(2016) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The minimal structure for iodotyrosine deiodinase function is defined by an outlier protein from the thermophilic bacterium Thermotoga neapolitana.

  Sun Z., Xu B., Spisak S., Kavran J.M., Rokita S.E.

  The nitroreductase superfamily of enzymes encompasses many flavin mononucleotide (FMN)-dependent catalysts promoting a wide range of reactions. All share a common core consisting of an FMN-binding domain, and individual subgroups additionally contain one to three sequence extensions radiating from ... >> More

  The nitroreductase superfamily of enzymes encompasses many flavin mononucleotide (FMN)-dependent catalysts promoting a wide range of reactions. All share a common core consisting of an FMN-binding domain, and individual subgroups additionally contain one to three sequence extensions radiating from defined positions within this core to support their unique catalytic properties. To identify the minimum structure required for activity in the iodotyrosine deiodinase subgroup of this superfamily, attention was directed to a representative from the thermophilic organism Thermotoga neapolitana (TnIYD). This representative was selected based on its status as an outlier of the subgroup arising from its deficiency in certain standard motifs evident in all homologues from mesophiles. We found that TnIYD lacked a typical N-terminal sequence and one of its two characteristic sequence extensions, neither of which was found to be necessary for activity. We also show that TnIYD efficiently promotes dehalogenation of iodo-, bromo-, and chlorotyrosine, analogous to related deiodinases (IYDs) from humans and other mesophiles. In addition, 2-iodophenol is a weak substrate for TnIYD as it was for all other IYDs characterized to date. Consistent with enzymes from thermophilic organisms, we observed that TnIYD adopts a compact fold and low surface area compared with IYDs from mesophilic organisms. The insights gained from our investigations on TnIYD demonstrate the advantages of focusing on sequences that diverge from conventional standards to uncover the minimum essentials for activity. We conclude that TnIYD now represents a superior starting structure for future efforts to engineer a stable dehalogenase targeting halophenols of environmental concern. << Less

  J. Biol. Chem. 297:101385-101385(2021) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Expression of a soluble form of iodotyrosine deiodinase for active site characterization by engineering the native membrane protein from Mus musculus.

  Buss J.M., McTamney P.M., Rokita S.E.

  Reductive deiodination is critical for thyroid function and represents an unusual exception to the more common oxidative and hydrolytic mechanisms of dehalogenation in mammals. Studies on the reductive processes have been limited by a lack of convenient methods for heterologous expression of the a ... >> More

  Reductive deiodination is critical for thyroid function and represents an unusual exception to the more common oxidative and hydrolytic mechanisms of dehalogenation in mammals. Studies on the reductive processes have been limited by a lack of convenient methods for heterologous expression of the appropriate proteins in large scale. The enzyme responsible for iodide salvage in the thyroid, iodotyrosine deodinase, is now readily generated after engineering its gene from Mus musculus. High expression of a truncated derivative lacking the membrane domain at its N-terminal was observed in Sf9 cells, whereas expression in Pichia pastoris remained low despite codon optimization. Ultimately, the desired expression in Escherichia coli was achieved after replacing the two conserved Cys residues of the deiodinase with Ala and fusing the resulting protein to thioredoxin. This final construct provided abundant enzyme for crystallography and mutagenesis. Utility of the E. coli system was demonstrated by examining a set of active site residues critical for binding to the zwitterionic portion of substrate. << Less

  Protein Sci. 21:351-361(2012) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Iodotyrosine deiodinase is the first mammalian member of the NADH oxidase/flavin reductase superfamily.

  Friedman J.E., Watson J.A. Jr., Lam D.W., Rokita S.E.

  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 succ ... >> More

  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. << Less

  J Biol Chem 281:2812-2819(2006) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Crystal structure of iodotyrosine deiodinase, a novel flavoprotein responsible for iodide salvage in thyroid glands.

  Thomas S.R., McTamney P.M., Adler J.M., Laronde-Leblanc N., Rokita S.E.

  The flavoprotein iodotyrosine deiodinase (IYD) salvages iodide from mono- and diiodotyrosine formed during the biosynthesis of the thyroid hormone thyroxine. Expression of a soluble domain of this membrane-bound enzyme provided sufficient material for crystallization and characterization by x-ray ... >> More

  The flavoprotein iodotyrosine deiodinase (IYD) salvages iodide from mono- and diiodotyrosine formed during the biosynthesis of the thyroid hormone thyroxine. Expression of a soluble domain of this membrane-bound enzyme provided sufficient material for crystallization and characterization by x-ray diffraction. The structures of IYD and two co-crystals containing substrates, mono- and diiodotyrosine, alternatively, were solved at resolutions of 2.0, 2.45, and 2.6 A, respectively. The structure of IYD is homologous to others in the NADH oxidase/flavin reductase superfamily, but the position of the active site lid in IYD defines a new subfamily within this group that includes BluB, an enzyme associated with vitamin B(12) biosynthesis. IYD and BluB also share key interactions involving their bound flavin mononucleotide that suggest a unique catalytic behavior within the superfamily. Substrate coordination to IYD induces formation of an additional helix and coil that act as an active site lid to shield the resulting substrate.flavin complex from solvent. This complex is stabilized by aromatic stacking and extensive hydrogen bonding between the substrate and flavin. The carbon-iodine bond of the substrate is positioned directly over the C-4a/N-5 region of the flavin to promote electron transfer. These structures now also provide a molecular basis for understanding thyroid disease based on mutations of IYD. << Less

  J. Biol. Chem. 284:19659-19667(2009) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Iodotyrosine dehalogenase 1 (DEHAL1) is a transmembrane protein involved in the recycling of iodide close to the thyroglobulin iodination site.

  Gnidehou S., Caillou B., Talbot M., Ohayon R., Kaniewski J., Noel-Hudson M.-S., Morand S., Agnangji D., Sezan A., Courtin F., Virion A., Dupuy C.

  In the thyroid, iodotyrosine dehalogenase acts on the mono and diiodotyrosines released during the hydrolysis of thyroglobulin to liberate iodide, which can then reenter the hormone-producing pathways. It has been reported that the deiodination of iodotyrosines occurs predominantly in the microsom ... >> More

  In the thyroid, iodotyrosine dehalogenase acts on the mono and diiodotyrosines released during the hydrolysis of thyroglobulin to liberate iodide, which can then reenter the hormone-producing pathways. It has been reported that the deiodination of iodotyrosines occurs predominantly in the microsomes and is mediated by NADPH. Recently, two cDNAs, 7401- and 7513-base pairs long that encode proteins with a conserved nitroreductase domain were published in GenBank as iodotyrosine dehalogenase 1 (DEHAL1) and iodotyrosine dehalogenase 1B (DEHAL1B), respectively. We report here our investigation of the localization and activity of one of these isoforms, DEHAL1. DEHAL1 mRNA is highly expressed in the thyroid, is up-regulated by cAMP, and encodes a transmembrane protein that efficiently catalyzes the NADPH-dependent deiodination of mono (L-MIT) and diiodotyrosine (L-DIT), with greater activity vs. L-MIT. Iodotyrosine deiodinase was active in HEK293 cells transfected by DEHAL1 cDNA, but not in CHO cells. A fraction of DEHAL1 protein is exposed to the cell surface, as indicated by biotinylation experiments. Immunohistochemistry studies showed that DEHAL1 proteins accumulate at the apical pole of thyrocytes. Taken together, these findings indicate that the deiodination reaction occurs at the apical pole of the thyrocyte and is involved in a rapid iodide recycling process at and/or close to the organification site. << Less

  FASEB J. 18:1574-1576(2004) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Purification of iodotyrosine deiodinase from bovine thyroid.

  Rosenberg I.N.

  Metabolism 19:785-798(1970) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Mutations in the iodotyrosine deiodinase gene and hypothyroidism.

  Moreno J.C., Klootwijk W., van Toor H., Pinto G., D'Alessandro M., Leger A., Goudie D., Polak M., Grueters A., Visser T.J.

  DEHAL1 has been identified as the gene encoding iodotyrosine deiodinase in the thyroid, where it controls the reuse of iodide for thyroid hormone synthesis. We screened patients with hypothyroidism who had features suggestive of an iodotyrosine deiodinase defect for mutations in DEHAL1. Two missen ... >> More

  DEHAL1 has been identified as the gene encoding iodotyrosine deiodinase in the thyroid, where it controls the reuse of iodide for thyroid hormone synthesis. We screened patients with hypothyroidism who had features suggestive of an iodotyrosine deiodinase defect for mutations in DEHAL1. Two missense mutations and a deletion of three base pairs were identified in four patients from three unrelated families; all the patients had a dramatic reduction of in vitro activity of iodotyrosine deiodinase. Patients had severe goitrous hypothyroidism, which was evident in infancy and childhood. Two patients had cognitive deficits due to late diagnosis and treatment. Thus, mutations in DEHAL1 led to a deficiency in iodotyrosine deiodinase in these patients. Because infants with DEHAL1 defects may have normal thyroid function at birth, they may be missed by neonatal screening programs for congenital hypothyroidism. << Less

  N. Engl. J. Med. 358:1811-1818(2008) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• A mammalian reductive deiodinase has broad power to dehalogenate chlorinated and brominated substrates.

  McTamney P.M., Rokita S.E.

  Iodotyrosine deiodinase is essential for iodide homeostasis and proper thyroid function in mammals. This enzyme promotes a net reductive deiodination of 3-iodotyrosine to form iodide and tyrosine. Such a reductive dehalogenation is uncommon in aerobic organisms, and its requirement for flavin mono ... >> More

  Iodotyrosine deiodinase is essential for iodide homeostasis and proper thyroid function in mammals. This enzyme promotes a net reductive deiodination of 3-iodotyrosine to form iodide and tyrosine. Such a reductive dehalogenation is uncommon in aerobic organisms, and its requirement for flavin mononucleotide is even more uncommon in catalysis. Reducing equivalents are now shown to transfer directly from the flavin to the halogenated substrate without involvement of other components typically included in the standard enzymatic assay. Additionally, the deiodinase has been discovered to act as a debrominase and a dechlorinase. These new activities expand the possible roles of flavin in biological catalysis and provide a foundation for determining the mechanism of this unusual process. << Less

  J. Am. Chem. Soc. 131:14212-14213(2009) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.