Cloning and Functional Characterization of Human Selenophosphate Synthetase, an Essential Component of Selenoprotein Synthesis

doi:10.1074/jbc.270.37.21659

Cloning and Functional Characterization of Human Selenophosphate Synthetase, an Essential Component of Selenoprotein Synthesis (*)

From the Thyroid Division, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

^§To whom correspondence should be addressed:
Thyroid Div., Brigham and Women's Hospital, 75 Francis St., Boston, MA 02115.
Tel.: 617-732-6737; Fax: 617-731-4718.

Abstract

Selenocysteine is co-translationally incorporated into prokaryotic and eukaryotic selenoproteins at in-frame UGA codons. However, the only component of the eukaryotic selenocysteine incorporation machinery identified to date is the selenocysteine-specific tRNA. In prokaryotes, selenocysteine is synthesized from seryl-tRNA and the active selenium donor, selenophosphate. Selenophosphate is synthesized from selenide and ATP by the selD gene product, selenophosphate synthetase, and is required for selenocysteine synthesis and incorporation into bacterial selenoproteins. We have now cloned human selD and shown that transfection of the human selD cDNA into mammalian cells results in increased selenium labeling of a mammalian selenoprotein, type 1 iodothyronine deiodinase. Despite significant differences between the mechanisms of selenoprotein synthesis in prokaryotes and eukaryotes, human selD weakly complements a bacterial selD mutation, partially restoring selenium incorporation into bacterial selenoproteins. Human selenophosphate synthetase has only 32% homology with the bacterial protein, although a highly homologous region that has similarity to a consensus ATP/GTP binding domain has been identified. Point mutations within this region result in decreased incorporation of selenium into type 1 iodothyronine deiodinase in all but one case. Further analysis revealed that reduced selenium labeling was due to altered ATP binding properties of the mutant selenophosphate synthetases.

Footnotes

↵* This work was supported by National Institutes of Health Grant DK47320 and a personal fellowship from the European Molecular Biology Organization (to S. C. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U34044[GenBank].
↵¹M. J. Berry, unpublished observations.
↵² The abbreviation used is:

Tricine

N-[2-hydroxy-1,1,bis(hydroxymethyl)ethyl]glycine.
- Received April 6, 1995.
- Revision received June 26, 1995.