Biochemical and structural investigation of Naegleria gruberi selenophosphate sintetase enzyme

Abstract

It has been the interest of many research groups to study the processes of translation of the genetic code into proteins that involve the synthesis of the amino acids Pirrolisin and Selenocysteine. Knowing that selenocysteine represents the major biological form of selenium, which is an essential micronutrient, and has specific synthesis pathways for bacteria, archaea and eukaryotes, studies are required in order to assess their particular evolutionary consequence and potential practical use. In the quest for elucidating the biological, chemical and structural properties of the 21st amino acid selenocysteine (Sec, U), it becomes a research emphasis the understanding of this route in Naegleria gruberi. Recently, the presence of the biosynthesis and incorporation of selenocisteínas in N. gruberi was described, and among the genes identified the Selenophosphate syntethase (SPS) plays a central role in the pathway. Involved in the catalysis of the conversion of selenide and adenosine 5'-triphosphate (ATP) in selenophosphate necessary for the synthesis of selenocysteine along with orthophosphate and adenosine 5'-monophosphate (AMP). N. gruberi SPS contains two distinct domains: the C-terminal domain, which has high identity with bacterial SPSs and an N-terminal domain, similar to methyltransferases from prokaryotes. Considering the evolutionary relationships with other basal eukaryotes and prokaryotes, this project seeks to study each domain separately on the atomic structure and physiological role in the cell. Immunochemical and biophysical characterizations are been implemented by native gel electrophoresis and Small Angle X-ray scattering (SAXS) as well as the screening of crystallization conditions. Together, these experiments may contribute to the understanding of the biosynthesis of selenocysteine in ancient eukaryotes.