Characterization of a Flavoprotein Iodotyrosine Deiodinase Suitable for Expression in Bacteria

Iodide salvage is essential to thyroid hormone metabolism and metabolic regulation. In mammals, the DEHAL1 gene product iodotyrosine deiodinase (IYD) is responsible for deiodination of the mono‐ and diiodotyrosine byproducts of thyroid hormone synthesis (triiodothyronine and thyroxine, T3 and T4, respectively). IYD is a membrane‐bound flavoprotein comprised of three domains with the catalytic domain belonging to the NADH oxidase/flavin reductase structural superfamily. This enzyme has been expressed in four systems: mammalian, insect, yeast, and bacterial cell lines. Mammalian and insect cell lines were expressed with the native gene sequence while yeast required codon optimization, and bacteria required additional engineering. Expressed IYD was characterized using CD spectra, kinetic rate constants, binding constants of substrates, and a series of crystal structures. Analysis of the crystal structure of IYD indicates a dimer with an active site comprising of both monomers and orienting the C‐I bond of iodotyrosine substrate stacking above the N5 of the flavin mononucleotide (FMN) required for activity. Three amino acids (E153, Y157, and K178) in the active site form hydrogen bonding and electrostatic contacts with the zwitterionic portion of the substrate. Mutation to any of these three amino acids significantly decreases substrate‐binding affinity and enzymatic activity.