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.
Highlights
The dehalogenation reaction catalyzed by IYD is unusual for aerobic organisms since the carbon-iodine bond is broken through a reductive process
Our laboratory has proposed a mechanism for IYD that is related to the catalytic strategy used by tetrachlorohydroquinone dehalogenase and iodothyronine deiodinase (ID) [21]
We report purification of IYD after its proteolytic release from porcine microsomes and expression of a homologous protein from mouse in human embryonic kidney (HEK) 293 cells
Summary
The dehalogenation reaction catalyzed by IYD is unusual for aerobic organisms since the carbon-iodine bond is broken through a reductive process. The best characterized examples of reductive dehalogenation in aerobic organisms are tetrachlorohydroquinone dehalogenase from Sphingomonas chlorophenolica [13, 14] and iodothyronine deiodinase (ID) from a variety of higher organisms including human [15]. Both enzymes catalyze reduction via similar chemical strategies. ID appears to act through an analogous tautomerization of thyroxin (3-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodophenylalanine) and transient oxidation of an active-site selenocysteine to yield the reduced product, triiodothyronine (16 –18) Both of these enzymes use the reducing power of exogenous thiols, and both contain a nucleophilic and redox-active residue in their active sites. Database analysis identifies IYD as the first mammalian representative of the NADH oxidase/flavin reductase superfamily, and quite distinct from mammalian iodothyronine dehalogenases [20, 22, 23]
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