Insight into the Structure and Mechanism of Iodotyrosine Deiodinase, the First Mammalian Member of the NADH Oxidase / Flavin Reductase Superfamily

Abstract

Iodotyrosine deiodinase (IYD) has remained poorly characterized for nearly 50 years in spite of its function as an iodine salvage pathway and its role in regulation of mammalian basal metabolism. IYD catalyzes the reductive deiodination of both mono- and diiodotyrosine, the iodinated side-products of thyroid hormone production. The Rokita lab previously purified IYD from porcine thyroids and identified a putative dehalogenase cDNA.

The work in this dissertation confirms the identity of the cDNA that encodes IYD through expression in HEK293 cells (KM = 4.4 ± 1.7 μM and Vmax = 12 ± 1 nmol hr-1 g-1) and, furthermore, identifies IYD as the first mammalian member of the NADH oxidase/flavin reductase superfamily, a protein fold previously found only in bacteria. In addition, a three-dimensional model of the NADH oxidase/flavin reductase domain of IYD was constructed based on the x-ray crystal structure coordinates (Protein Data Bank code 1ICR) of the minor nitroreductase from Escherichia coli. The model also predicts structural features of IYD, including interactions between the flavin bound to IYD and one of three conserved cysteines.

To investigate the role of the NADH oxidase/flavin reductase domain plays in electron transfer, two truncation mutants were generated: IYD-NR (residues 81-285) and IYD-ΔTM (residues 34-285) encoding transmembrane-domain deleted IYD. The two mutants were expressed in HEK293 cells and their catalytic properties were measured. IYD-NR did not promote deiodination (Vmax = 0.0 ± 0.4 nmol hr-1 ug-1) of diiodotyrosine in a literature-derived iodide release assay. However, IYD-ΔTM was catalytically active toward DIT (KM = 4.6 ± 1.3 μM and Vmax = 7.0 ± 0.5 nmol hr-1 g-1) when the chemical reductant dithionite was used but did not promote NADPH-responsive deiodination. The subcellular location for both mutants was determined by ultracentrifugation. IYD-NR was observed in the insoluble fraction after centrifugation at 100,000 x g. However, IYD-ΔTM remained in the supernatant after centrifugation at 100,000 x g for 1 hour.