Endocrine Abstracts (2019) 63 OC9.2 | DOI: 10.1530/endoabs.63.OC9.2

Direct evidence for disulfides in the mechanism of deiodinase 1

Alfonso Rubén Ruiz Rodriguez, Doreen Braun & Ulrich Schweizer


Rheinische Friedrich Wilhelms Universität Bonn, Bonn, Germany.


The main product of the thyroid gland, T4, is a prohormone. 5’-deiodination is needed to convert T4 into the active, nuclear receptor-binding hormone, T3. Pharmacological modulation of activating 5’-and inactivating 5-deiodination of iodothyronines would be desirable in a number of medical conditions, including hypothyroidism, hyperthyroidism and various cancers. A structural and mechanistic understanding of deiodinase (DIO) catalysis is a prerequisite of successful pharmacological intervention. Structural data on deiodinases are currently limited on the intracellular catalytic domain of murine Dio3 (1). However, deiodinases are membrane anchored proteins and it is known that the membrane anchor contributes to activity of the enzymes. In order to elucidate the mechanism of deiodinases, we have recombinantly expressed full length human DIO1 as a membrane protein in insect cells. Since these cells do not have the capacity to express selenoproteins, selenocysteine (Sec)126 was replaced by cysteine (Cys). The resulting protein was expressed at high levels and catalytically active in the presence of mM concentrations of DTT as reductant. Similar to murine Dio3, human DIO1 was active in the presence of a physiological reducing system comprising of glutaredoxin (1 μM), glutathione (GSH), GSH reductase, and NADPH. We replaced a series of conserved Cys in DIO1 with Ser or Ala and showed that all these mutations reduced the activity of the enzyme. We could assign different functions to these cysteines. DIO1 is known to function as a dimer. A new finding is that according to mass-spec analyses, Cys95 and possibly Cys105 contribute to stabilization of the dimer via inter-subunit disulfides. In addition, mass-spec analysis for the first time directly demonstrated the formation of a disulfide between Cys124 and Cys(Sec)126 during catalysis as suggested before. Data derived from analysis of mutant DIO1 are compatible with a reducing mechanism similar to that in murine Dio3. Taken together we show for the first time the formation of inter-subunit and intra-subunit disulfides within DIO1. Their function is revealed by the use of a physiological reducing system comprised of Grx, GSH, GR, and NADPH instead of the non-physiological provision of the synthetic DTT reductant.

Reference: 1. Schweizer U, Schlicker C, Braun D, Köhrle J, Steegborn C. (2014) Crystal structure of mammalian selenocysteine-dependent iodothyronine deiodinase suggests a peroxiredoxin-like catalytic mechanism. Proc Natl Acad Sci U S A. 2014 Jul 22;111(29):10526–31. doi: 10.1073/pnas.1323873111.

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