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

Abstract

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.

FIGURE 1.

Reductive deiodination for iodide salvage catalyzed by IYD (A) and metabolism of the hormone thyroxine by iodothyronine deiodinase (ID) (B).

FIGURE 2.

IYD structure. A, an overall view of the native homodimer of IYD crystallized in the absence of substrate. Each monomer is distinguished by color. Disordered regions consisting of residues 156–177 and 195–208 connect to the structure as indicated by ★ and ☆, respectively. B, native homodimer of IYD crystallized in the presence of its substrate, MIT. Only the structure induced upon substrate binding is highlighted in the colors of the monomers shown in A. The surface properties of IYD (C) and its complex with monoiodotyrosine (D) were calculated using vacuum electrostatics in PyMOL (47). Blue indicates positive charge, and red indicates negative charge. E, ionic interactions and hydrogen bonding stabilize the FMN·monoiodotyrosine complex formed by IYD. F, the interaction between FMN and MIT in the active site of IYD. An F_o − F_c electron density map calculated after refinement in the absence of FMN and MIT is shown contoured at 3σ.

FIGURE 3.

Alignment of secondary structure for representatives of the NADH oxidase/flavin reductase superfamily. IYD and BluB now define a third subclass of the NADH oxidase/flavin reductase superfamily. NADH oxidase (NOX) and FRP illustrate the α-β fold for the original two subclasses of this superfamily. The boxed regions indicate the sequences that form the active site lids. The dotted lines indicate spacing inserted for alignment. Structural assignments were derived from crystallographic data (Protein Data Bank codes 3GFD, 2ISL, 1NOX, and 2BKJ, respectively).

FIGURE 4.

Conformational changes in the IYD active site to accommodate MIT and DIT. A, the surface characteristics of the active site of IYD for the IYD·MIT co-crystal crystal calculated using vacuum electrostatics in PyMOL (47). Blue indicates positive charge, and red indicates negative charge. B, alignment of active site structures of IYD bound with MIT (orange) and DIT (cyan) illustrates the minor conformational change required to accommodate the larger substrate.

FIGURE 5.

Polar contacts between bound FMN and IYD (A), BluB (B), and FRP (C). Coordination of FMN by protein is highlighted for residues within 4 Å of the flavin in the crystal structures for Protein Data Bank codes 3GDF, 2ISJ, and 2BKJ, respectively.

FIGURE 6.

Structural overlay of IYD and BluB. Structural differences between IYD·MIT (gray) and BluB (blue) are highlighted using an overlaid stereoimage. Structural changes near the active site are depicted with the schematic representation. The full-view overlay can be seen as a stereoimage in the supplemental information.

FIGURE 7.

Mapping human mutations onto the structure of IYD. Native residues of IYD (M. musculus) highlighted in red correspond to sites associated with human mutations identified clinically to cause hypothyroidism (1). Other color coding is consistent with the previous illustrations (see legends) and distinguishes the two subunits within the dimer and the FMN.