The distribution and mechanism of iodotyrosine deiodinase defied expectations

Abstract

Iodotyrosine deiodinase (IYD) is unusual for its reliance on flavin to promote reductive dehalogenation under aerobic conditions. As implied by the name, this enzyme was first discovered to catalyze iodide elimination from iodotyrosine for recycling iodide during synthesis of tetra- and triiodothyronine collectively known as thyroid hormone. However, IYD likely supports many more functions and has been shown to debrominate and dechlorinate bromo- and chlorotyrosines. A specificity for halotyrosines versus halophenols is well preserved from humans to bacteria. In all examples to date, the substrate zwitterion establishes polar contacts with both the protein and the isoalloxazine ring of flavin. Mechanistic data suggest dehalogenation is catalyzed by sequential one electron transfer steps from reduced flavin to substrate despite the initial expectations for a single two electron transfer mechanism. A purported flavin semiquinone intermediate is stabilized by hydrogen bonding between its N5 position and the side chain of a Thr. Mutation of this residue to Ala suppresses dehalogenation and enhances a nitroreductase activity that is reminiscent of other enzymes within the same structural superfamily.

Keywords: Dehalogenase; Flavin; Iodide salvage; Reductive dehalogenation; Thyroid.

Figure 1

Conservation of key residues coordinating substrate, protein and FMN. (A) Active site residues illustrated by human IYD (PDB 4TTC) form polar interactions over distances ranging from architecture of this family is easily detected.

Figure 2

Sequence distribution in the active site lid of IYD responsible for substrate recognition.

Figure 3

Sequence domains of IYD.

Figure 4

Crystal structures of human IYD containing FMN_ox in the absence and presence of I-Tyr. The green and cyan ribbons represent the two identical polypeptides that assemble into an active dimer. (A) In the absence of substrate, the active site lid (helix turn helix, residues 161–178) and loop (199–211) do not adopt a single stable conformation (PDB 4TTB) [31]. (B) In the presence of I-Tyr, the lid and loop form a stable and detectable structure that surrounds I-Tyr and collapses over the active site (PDB 4TTC) [31]. Carbon atoms of FMN are colored orange and carbons in the I-Tyr are colored yellow (and shown as spheres).

Figure 5

Structural diversity in the nitro-FMN superfamily is greatest in the active site domain. The α-β dimeric core regions of IYD (PDB 4TTC) [31], flavin reductase (FRP, PDB 2BKJ [41]), nitroreductase (NfsB, PDB 1YKI [46]) and flavin destructase (BluB, PDB 2ISJ [40]) are shown in gray. The variable active site regions are illustrated in green for IYD, cyan for BluB, magenta for FRP and yellow for NfsB. For simplicity, the C-terminal extension (~ 50 residues) of FRP is omitted and the carbon atoms of FMN of are shown in gray.

Figure 6

Polar coordination to the isoalloxazine ring of FMN in enzymes of the nitro-FMN reductase superfamily. Carbons of the protein side chains and FMN are illustrated in cyan and orange, respectively. Carbons of I-Tyr are illustrated in yellow. Coordination is illustrated for (A) IYD (PDB 4TTC [31]), (B) flavin destructase BluB (PDB 2ISJ [40]), (C)

Figure 7

Redox titration of IYD in the absence and presence of an inert substrate analog F-Tyr [31].

Scheme 1

Two different types of enzymes promote the unusual process of reductive

Scheme 2

Proposed mechanism of dehalogenation involving successive one electron transfers by FMN.

Scheme 3

A pyridone-based derivative of Tyr mimics the keto form of substrates for IYD.

Scheme 4

One electron donors promote reductive dehalogenation of α-halo ketones [84].

Scheme 5

The redox characteristics of FMN control dehalogenation versus nitroaromatic reduction [36].