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Insights into the Mechanism of Human Deiodinase 1

Title data

Rodriguez-Ruiz, Alfonso ; Braun, Doreen ; Pflug, Simon ; Brol, Alexander ; Sylvester, Marc ; Steegborn, Clemens ; Schweizer, Ulrich:
Insights into the Mechanism of Human Deiodinase 1.
In: International Journal of Molecular Sciences. Vol. 23 (2022) Issue 10 . - No. 5361.
ISSN 1422-0067
DOI: https://doi.org/10.3390/ijms23105361

Abstract in another language

The three isoenzymes of iodothyronine deiodinases (DIO1-3) are membrane-anchored homo-dimeric selenoproteins which share the thioredoxin-fold structure. Several questions regarding their catalytic mechanisms still remain open. Here, we addressed the roles of several cysteines which are conserved among deiodinase isoenzymes and asked whether they may contribute to dimerization and reduction of the oxidized enzyme with physiological reductants. We also asked whether amino acids previously identified in DIO3 play the same role in DIO1. Human DIO1 and 2 were recombinantly expressed in insect cells with selenocysteine replaced with cysteine (DIO1U126C) or in COS7 cells as selenoprotein. Enzyme activities were studied by radioactive deiodination assays with physiological reducing agents and recombinant proteins were characterized by mass spectrometry. Mutation of Cys124 in DIO1 prevented reduction by glutathione, while 20 mM dithiothreitol still regenerated the enzyme. Protein thiol reductants, thioredoxin and glutaredoxin, did not reduce DIO1U126C. Mass spectrometry demonstrated the formation of an intracellular disulfide between the side-chains of Cys124 and Cys(Sec)126. We conclude that the proximal Cys124 forms a selenenyl-sulfide with the catalytic Sec126 during catalysis, which is the substrate of the physiological reductant glutathione. Mutagenesis studies support the idea of a proton-relay pathway from solvent to substrate that is shared between DIO1 and DIO3.

Further data

Item Type: Article in a journal
Refereed: Yes
Keywords: kinetic analysis; mass spectrometry; selenocysteine; selenoprotein; thyroid hormone
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry > Chair Biochemistry - Univ.-Prof. Dr. Clemens Steegborn
Result of work at the UBT: Yes
DDC Subjects: 500 Science > 540 Chemistry
500 Science > 570 Life sciences, biology
Date Deposited: 10 Nov 2022 08:41
Last Modified: 10 Nov 2022 08:41
URI: https://eref.uni-bayreuth.de/id/eprint/72733