Selenium is mobilized in vivo from free selenocysteine and is incorporated specifically into formate dehydrogenase H and tRNA nucleosides.

Selenophosphate synthetases (SPS) from Escherichia coli (18) and the closely related enzyme from Haemophilus influenzae (7) have been characterized. In the presence of high levels of free selenide (1 to 5 mM) and dithiothreitol (8 to 10 mM) that are included in the in vitro assay system, the apparent Km values for ATP and selenide are 1 mM and 20 μM, respectively. A Km value of 20 μM for selenide is far above the optimal concentration (0.1 to 1 μM) needed for the growth of various bacterial species and cultured mammalian cells. In fact, levels above 10 μM are toxic for many bacterial species. Considering that intracellular concentrations of selenium should not approach toxic levels, a mechanism to provide SPS with adequate amounts of selenium may be necessary. Genetic analysis of iron-sulfur cluster formation in the nitrogenase protein from Azotobacter vinelandii led to the identification of a pyridoxal 5′-phosphate-dependent protein. This protein, referred to as NifS, catalyzed the desulfurization of cysteine to alanine and a sulfur transfer form, referred to as S* (19). (The products of the cysteine desulfurase and selenocysteine lyase reactions have been referred to in the literature as elemental sulfur [S0] and elemental selenium [Se0]. Designation of the product as S0 or Se0 implies that the redox state of the product is known. Since the redox state has not been determined, it is now proposed by Klaus-Dieter Asmus of Notre Dame University that both products can be designated as a sulfur transfer form [S*] and as a selenium transfer form [Se*] until a redox state is determined.) In vitro experiments performed with an inactive apo-Fe nitrogenase demonstrated that the NifS protein and l-cysteine could replace high concentrations of sulfide required for the reconstitution of the iron-sulfur cluster in nitrogenase, which resulted in a recovery of 85 to 90% of the original enzymatic activity (20). NifS homologs have since been identified in both prokaryotes and eukaryotes. The cysteine desulfurase activity is essential in the delivery of sulfur for iron-sulfur cluster formation (18) and the biosynthesis of thiamine, biotin, and 4-thiouridine (s4U) in tRNA (9). In addition to cysteine, NifS-like proteins also act on selenocysteine as the substrate. Members of a similar family of proteins, the selenocysteine lyase (SCL) enzymes, resemble NifS in both structure and function (2, 3). SCL enzymes catalyze the pyridoxal 5′-phosphate-dependent decomposition of selenocysteine to alanine and a selenium transfer form referred to as Se*. SCL enzymes exhibit high specificity towards selenocysteine with little or no reactivity towards cysteine. The chemical similarity between selenium and sulfur allows selenium to enter the bacterial metabolism via the cysteine biosynthetic pathway, where it can be incorporated into free selenocysteine and selenomethionine (14). Selenocysteine can be inserted into proteins nonspecifically, or a lyase can utilize it to generate selenium. The possibility that SCL enzymes can function as selenium delivery proteins for SPS has been considered (6, 8) The generation of selenium near SPS may be an attractive solution to the obstacle of selenide toxicity and the high Km value for selenide exhibited by SPS. Recent in vitro experiments demonstrated that the NifS protein from A. vinelandii (6) and the three NifS-like proteins from E. coli (8) can effectively mobilize selenium from free selenocysteine to SPS as a substrate. In fact, determined SPS activity was greater than with selenide alone as the substrate. In the present study, in vivo evidence is presented to support the participation of the E. coli NifS-like proteins as components of a selenium delivery system for the biosynthesis of selenophosphate.