Crystal Structure of Iodotyrosine Deiodinase, a Novel Flavoprotein Responsible for Iodide Salvage in Thyroid Glands

Seth R Thomas,Patrick M Mctamney,Jennifer M Adler,Nicole Laronde-Leblanc,Steven E Rokita

doi:10.1074/jbc.m109.013458

Seth R Thomas, Patrick M Mctamney + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m109.013458

Copy DOI

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.

Highlights

Tem is accomplished by a Naϩ/IϪ symporter located in the plasma membrane of thyroid follicular cells [2]
Flavin has been implicated in just one bacterial reductive dehalogenation, little information is available on this additional system other than its use of flavin-adenine dinucleotide (FAD) rather than flavin mononucleotide (FMN) [12]
The resulting protein was screened for crystallization, and crystals were obtained for iodotyrosine deiodinase (IYD) alone and IYD containing its substrates MIT and DIT alternatively

Summary

EXPERIMENTAL PROCEDURES

Method using two parts IYD (16 mg/ml; 10 mM potassium phosphate, pH 7.4) to one part reservoir solution. Sf9 cells adapted to SF-900 II SFM (Invitrogen) were trans- for IYD(⌬TM)His was collected at a wavelength of 1.653 Å fected with the pFASTBAC1-IYD(⌬TM)His recombinant for single anomalous diffraction phasing using sulfur atoms. The resulting Diffraction data of co-crystals containing MIT and DIT recombinant baculovirus was collected from the growth medium alternatively were collected at 0.9795 Å. Plicity of infection of 0.05 (plaque-forming unit/cell) as directed by Phasing and Refinement—All nine sulfur positions within the Bac-to-Bac protocols, and virus concentration was determined IYD(⌬TM)His were found using HKL2MAP [15]. One dimer of this co-crystal was used in turn as the model through a 0.22-␮m membrane and loaded onto a HisTrap for molecular replacement to solve the co-crystal containing HP column (1 ml) with chelated Ni2ϩ.

Protein Data Bank code

RESULTS

DISCUSSION