Selenium is an essential nutrient that generally occurs as a component of several prokaryotic and eukaryotic proteins as a selenocysteine moiety. The incorporation of selenocysteine in prokaryotes requires selenophosphate as a reactive selenium donor. Selenophosphate synthetase from Escherichia colii catalyzes the synthesis of selenophosphate, AMP, and orthophosphate from selenide and ATP. The enzyme mechanism is believed to proceed through an enzyme-pyrophosphoryl intermediate; however, experiments designed to prove this have been inconclusive. To help elucidate the mechanism, the enzyme has been extensively purified for use in isotope studiesabeled ATP. These experiments will be carried out in the laboratory of Dr. Frank Raushel at Texas A&M University. Furthermore, highly purified enzyme will also be used in attempts to crystallize the enzyme for three-dimensional structure determination. In order to obtain an enzyme form that may be more amenable to mechanistic studies, we have attempted to purify the enzyme from alternate sources. Current purification experiments are being carried out with extracts from Methanoccus vannielii. Based on Western blot analysis, this organism appears to be a more abundant source of the enzyme. As an archaebacteria, the enzyme from this organism may differ from both eukaryotic and prokaryotic forms, possibly making it more suitable for mechanistic studies. Two different proteins that cross- react with antibodies to the E. coli protein have been partially purified from M. vannielii using standard techniques. Based on the sequence of the first 50 amino acids, the first protein was found to be almost identical to N5, N10-methylenetetrahydromethanopterin reductase from Methanobacterium thermoautotrophicum. The second cross-reacting protein showed no homology to other known proteins in the first 10 amino acid residues. However, selenophosphate synthetase activity was detected during the early stages of the purification.
