Engineering the elongation factor Tu for efficient selenoprotein synthesis.
Bottom Line: Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis.Selection was carried out for enhanced Sec incorporation into hAGT; the resulting EF-Tu variants contained highly conserved amino acid changes within members of the library.The improved UTu-system with EF-Sel1 raises the efficiency of UAG-specific Sec incorporation to >90%, and also doubles the yield of selenoprotein production.
Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.Show MeSH
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Mentions: The opportunity to make active Sec-containing hAGT variants led us to engage in a short mechanistic study. The accepted reaction mechanism of the hAGT protein requires the C145 thiol group to be assisted by nearby residues for deprotonation in order to form the active thiolate that then nucleophilically attacks the alkyl group of O6-alkylguanine in DNA (16). It was proposed (16) that the H146-E172 dyad deprotonates the C145 thiol group through a bridging water molecule (Figure 4). Additionally, the C145 thiol group is in direct contact with N137, but the function of this residue was unclear (16). We felt that a comparison of the properties of the hAGT Cys145 and Sec145 enzymes should clarify the hAGT mechanism. With a pKa of 5.5 Sec is present under physiological conditions as a selenolate, while Cys (pKa is 8.6) is a thiol. Guided by the structures (16,17), hAGT mutants in positions 24, 137, 146 and 172 were made. The individual variants C24A, N137A, H146N and E172A all contained the wild-type C145. Another set was created in which the variant had UAG145; this set was expected to show U insertion in the UTu system. Cells co-expressing the hAGT variants with the UTu-system in the presence of wild-type EF-Tu (without additional EF-Tu variants) were subjected to three rounds of MNNG selection before plating to reveal the surviving fractions (Figure 4). Surprisingly, the N146 mutant retained equal wild-type like activity for both hAGT C145 and U145 variants. In contrast, the A137 variant inactivated the hAGT C145 protein, but sustained the wild-type activity for the AGT U145 selenoprotein. Hence, the presence of N137 is important for hAGT/C145 activity, while the presence of H146 is not.
Affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.