Iodine is an essential trace element that plays a key role in the biosynthesis of the thyroid hormones thyroxine (T4) and triiodothyronine (T3). Their biosynthesis is based on the iodination of tyrosine residues and their subsequent oxidative coupling within the protein precursor thyroglobulin. Efficient hormonogenesis depends on the structural characteristics of hormonogenic sites, particularly the appropriate spatial arrangement and orientation of tyrosine residues and their local amino acid environment. Iodinated proteins and compounds resembling thyroid hormones have been identified in plants; however, the mechanisms underlying their formation remain unclear. Previous structural analyses of plant proteins have suggested that some may contain tyrosine residues with characteristics similar to the hormonogenic sites of thyroglobulin. One such candidate is the Rpb8b protein from Arabidopsis thaliana.
In the experimental part of this study, recombinant Rpb8b was used as a model system. The protein was expressed in a bacterial expression system, purified by affinity chromatography, and subjected to chemical and enzymatic iodination. Chemical iodination involved the use of an oxidizing agent to generate reactive iodine species, whereas enzymatic iodination was performed using a glucose oxidase–lactoperoxidase system. The effects of iodination were evaluated by SDS-PAGE and native PAGE.
We found that chemical iodination resulted in greater heterogeneity of protein forms and may have affected protein stability, whereas enzymatic iodination proceeded under more controlled reaction conditions and Rpb8b was better preserved. Changes following iodination were more pronounced under native PAGE conditions, suggesting that the modifications affected the charge and conformation of the protein. The results demonstrate that Rpb8b can be modified using both iodination approaches, with enzymatic iodination proving more suitable for further studies of the potential role of Rpb8b in the formation of T3 and T4.
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