Review
Selenocysteine in proteins—properties and biotechnological use

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Abstract

Selenocysteine (Sec), the 21st amino acid, exists naturally in all kingdoms of life as the defining entity of selenoproteins. Sec is a cysteine (Cys) residue analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys. The selenium atom gives Sec quite different properties from Cys. The most obvious difference is the lower pKa of Sec, and Sec is also a stronger nucleophile than Cys. Proteins naturally containing Sec are often enzymes, employing the reactivity of the Sec residue during the catalytic cycle and therefore Sec is normally essential for their catalytic efficiencies. Other unique features of Sec, not shared by any of the other 20 common amino acids, derive from the atomic weight and chemical properties of selenium and the particular occurrence and properties of its stable and radioactive isotopes. Sec is, moreover, incorporated into proteins by an expansion of the genetic code as the translation of selenoproteins involves the decoding of a UGA codon, otherwise being a termination codon. In this review, we will describe the different unique properties of Sec and we will discuss the prerequisites for selenoprotein production as well as the possible use of Sec introduction into proteins for biotechnological applications. These include residue-specific radiolabeling with gamma or positron emitters, the use of Sec as a reactive handle for electophilic probes introducing fluorescence or other peptide conjugates, as the basis for affinity purification of recombinant proteins, the trapping of folding intermediates, improved phasing in X-ray crystallography, introduction of 77Se for NMR spectroscopy, or, finally, the analysis or tailoring of enzymatic reactions involving thiol or oxidoreductase (redox) selenolate chemistry.

Section snippets

Selenium

The basic element selenium was discovered by the Swedish chemist Berzelius in 1817. It belongs to group VIA in the periodic table, which also includes oxygen, sulfur, and tellurium; three elements with which selenium thus shares many properties. Six stable isotopes and a number of radionuclides with different characteristics exist for selenium. Selenium indeed displays many similarities with sulfur, i.e. they have rather similar electronegativities and atom sizes and they have the same major

Selenoproteins

The importance of selenium as a trace element, being essential for mammals, is mainly due to vital functions of at least some selenoproteins. Selenoproteins contain selenium in the form of Sec, the 21st amino acid, being a Cys-analogue with a selenium atom replacing the sulfur atom in Cys. As discussed above, selenium and sulfur, while being related elements, differ in chemical properties. Thus, Sec residues exhibit different characteristics compared to a Cys residue (Table 2) and give unique

Sec-containing proteins versus the corresponding Cys homologs

The highly increased reactivity of selenoproteins compared to their Cys-dependent counterparts is generally regarded as an evolutionary pressure for the development of selenoproteins. The other point of view is that Sec would be an ancient amino acid, to a major extent lost during evolution due to its high reactivity. Whatever the true reason is for selenoprotein existence, it is clear that the insertion of a Sec residue in a protein constitutes a metabolically costly synthesis machinery (see

Production of selenoproteins

Synthetic production of selenoproteins is far from trivial because of the unique properties and high reactivity of Sec. On the other hand, if succeeded, it may have major biotechnological potential as a result of those properties. One method involves the production of recombinant selenoproteins in E. coli, as shall be discussed in further detail below. An alternative approach is to synthetically incorporate a Sec residue into a protein using different chemical substitution reactions. The first

Targeted selenocysteine incorporation at the ribosome—expanding the genetic code

A Sec residue of a selenoprotein is in Nature co-translationally incorporated at a specific predefined UGA codon. The UGA codon, which normally confers termination of translation by binding of a release factor, is re-coded to Sec-incorporation by species-specific mechanisms guided by a secondary structure in the mRNA. For E. coli, this mechanism has been thoroughly studied by Böck and coworkers mainly using synthesis of the selenoprotein formate dehydrogenase H as a model [64], [65]. In short,

Recombinant selenoprotein production in E. coli

The fact that mammalian selenoprotein genes are not compatible with the bacterial Sec incorporation machinery makes it impossible to directly heterologously express them as recombinant selenoproteins in E. coli. One possibility, as also listed in Table 4, is to use a cysteine auxotrophic E. coli strain, which allows the substitution of Cys to Sec at growth in the absence of sulfur and instead the presence of a selenium source [77]. This method is, however, not suitable if the protein of

Biotechnological use of Sec insertion

The possibility to introduce a Sec residue into proteins may be of substantial importance for a number of different applications. In addition to facilitating studies of natural selenoproteins, Sec insertion can also be used for different biotechnological applications in a number of research fields (see Table 5). We shall now briefly discuss these different applications. All of the methods are based on either the introduction of a selenium isotope, with specific characteristics such as

Conclusions

Research regarding the 21st amino acid, selenocysteine, has, during the last 30 years, progressed from an intriguing finding of Sec as part of a few selected proteins, to the recognition of Sec as an essential component of many living organisms, coupled to human disorders, and being translated by an expansion of the genetic code. The field of study on proteins naturally containing selenocysteine is rapidly growing, with new selenoproteins being found that await to be characterized. The

Acknowledgements

The research of the authors is supported by the Karolinska Institute, the Swedish Cancer Society, the Swedish Research Council for Medicine, and Lars Hiertas Foundation.

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