Search

The evolution of the genetic code to incorporate selenocysteine (Sec) enabled the development of a selenoproteome in all domains of life. O-phosphoseryl-tRNASec selenium transferase (SepSecS) catalyzes the terminal reaction of Sec synthesis on tRNASec in archaea and eukaryotes. Despite harboring four equivalent active sites, human SEPSECS binds no more than two tRNASec molecules. Though, the basis for this asymmetry remains poorly understood. In humans, an acidic, C-terminal, α-helical extension precludes additional tRNA-binding events in two of the enzyme monomers, stabilizing the SEPSECS•tRNASec complex. However, the existence of a helix exclusively in vertebrates raised questions about the evolution of the tRNA-binding mechanism in SEPSECS and the origin of its C-terminal extension. Herein, using a comparative structural and phylogenetic analysis, we show that the tRNA-binding motifs in SEPSECS are poorly conserved across species. Consequently, in contrast to mammalian SEPSECS, the archaeal ortholog cannot bind unacylated tRNASec and requires an aminoacyl group. Moreover, the C-terminal α-helix 16 is a mammalian innovation, and its absence causes aggregation of the SEPSECS•tRNASec complex at low tRNA concentrations. Altogether, we propose SEPSECS evolved a tRNASec binding mechanism as a crucial functional and structural feature, allowing for additional levels of regulation of Sec and selenoprotein synthesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Selenophosphate synthetase (SEPHS) plays an essential role in selenium metabolism. Two mammalian SEPHS paralogues, SEPHS1 and SEPHS2, share high sequence identity and structural homology with SEPHS. Here, we report nine individuals from eight families with developmental delay, growth and feeding problems, hypotonia, and dysmorphic features, all with heterozygous missense variants in SEPHS1. Eight of these individuals had a recurrent variant at amino acid position 371 of SEPHS1 (p.Arg371Trp, p.Arg371Gln, and p.Arg371Gly); seven of these variants were known to be de novo. Structural modeling and biochemical assays were used to understand the effect of these variants on SEPHS1 function. We found that a variant at residue Trp352 results in local structural changes of the C-terminal region of SEPHS1 that decrease the overall thermal stability of the enzyme. In contrast, variants of a solvent-exposed residue Arg371 do not impact enzyme stability and folding but could modulate direct protein-protein interactions of SEPSH1 with cellular factors in promoting cell proliferation and development. In neuronal SH-SY5Y cells, we assessed the impact of SEPHS1 variants on cell proliferation and ROS production and investigated the mRNA expression levels of genes encoding stress-related selenoproteins. Our findings provided evidence that the identified SEPHS1 variants enhance cell proliferation by modulating ROS homeostasis. Our study supports the hypothesis that SEPHS1 plays a critical role during human development and provides a basis for further investigation into the molecular mechanisms employed by SEPHS1. Furthermore, our data suggest that variants in SEPHS1 are associated with a neurodevelopmental disorder., Competing Interests: Declaration of interests S.V.M., E.T., H.G., T.B.P., A.T., D.A.C., M.M.M., I.M.W., K.G.M., and J.J. are employees of GeneDx., LLC. This article was prepared while M.S. was employed at the University of Illinois at Chicago. The opinions expressed in this article are the author’s own and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government., (Copyright © 2024 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)