The universal donor for specific selenium incorporation into proteins has been identified as monoselenophosphate. Selenophosphate synthetase catalyzes the formation of selenophosphate from selenide and ATP. The catalytic mechanism of the selenophosphate synthetase enzyme from E. coli has been studied in detail. The enzyme has a very low determined specific activity that may be due to the use of selenide as a substrate. The determined Km value for selenide is very high and approaches levels that are considered toxic to cell growth. It was recently demonstrated that the replacement of selenide in the in vitro selenophosphate synthetase assay with selenocysteine and the NIFS protein resulted in higher rates of selenophosphate formation. It was proposed that the use of a selenocysteine lyase protein may resemble a mechanism by which E. coli generates free selenide for selenophosphate synthetase. A recent collaboration was established with Professor Nobuyoshi Esaki at Kyoto University in Japan. Professor Esaki has cloned and overexpressed several NIFS-like proteins from E. coli. One protein, CSDB, has been characterized and described as a selenocysteine-specific lyase. The CSDB protein along with selenocysteine, in place of selenide, was added to the in vitro selenophosphate synthetase assay. CSDB proved to be very effective in generating selenide as a substrate for selenophosphate synthetase and, like the NIFS protein, its presence resulted in higher rates of selenophosphate formation. To better understand the interaction of CSDB with selenophosphate synthetase, an inactive mutant of CSDB, R379A, was used in competition experiments. The presence of increasing concentrations of R379A in the CSDB selenophosphate synthetase assays did not affect the rate of selenophosphate formation, suggesting the two proteins may not form a complex. Additionally, the H. influenzae selenophosphate synthetase protein, which contains a selenocysteine that aligns with cysteine 17 in the E. coli enzyme, was mutated to an arginine and threonine residue. These residues align with selenocysteine and are found in selenophosphate synthetase enzymes from drosophila and human sources. Both mutants were purified and their corresponding activity was determined with selenide as a substrate. The threonine mutant had low detectable activity and the arginine mutant had none. However, when both mutants were assayed in the presence of the NIFS protein and selenocysteine in place of selenide, both were active. Additional work is now being performed to better understand the role of selenocysteine lyase enzymes in the biosynthesis of selenophosphate. - selenophosphate, selenophosphate synthetase, selenide delivery protein
| Lacourciere, G M; Mihara, H; Kurihara, T et al. (2000) Escherichia coli NifS-like proteins provide selenium in the pathway for the biosynthesis of selenophosphate. J Biol Chem 275:23769-73 |
| Lacourciere, G M; Stadtman, T C (1999) Catalytic properties of selenophosphate synthetases: comparison of the selenocysteine-containing enzyme from Haemophilus influenzae with the corresponding cysteine-containing enzyme from Escherichia coli. Proc Natl Acad Sci U S A 96:44-8 |
| Lacourciere, G M (1999) Biosynthesis of selenophosphate. Biofactors 10:237-44 |
