Selenium has been reported to provide many health benefits in animals, including humans, when obtained from the diet in adequate amounts. For example, selenium has been known to play roles in cancer prevention, aging retardation, immune augmentation, prevention of heart diseases, muscle development and development [[1–4] and references therein]. Many of the health benefits of selenium are mediated by selenoproteins, which contain selenocysteine (Sec) as a selenium containing amino acid .
Selenophosphate synthetase (SPS) synthesizes selenophosphate (SeP), the active selenium donor in Sec biosynthesis, using selenide and ATP as substrates . SeP serves as a selenium donor during Sec biosynthesis . Sec is contained in all selenoproteins . SPS was first isolated from Escherichia coli as one of the enzymes involved in selenoprotein synthesis and was designated SelD . Only one type of SPS, SelD, exists in lower eukaryotes and eubacteria, however, there are two isoforms of SPS, SPS1 and SPS2, that occur in higher eukaryotes . One of the major differences in the sequences between SPS1 and SPS2 is that SPS1 has an arginine at the position corresponding to Sec in SPS2 .
Although it is not clear why there are two SPSs in higher eukaryotes, recent studies have shown that SPS2 synthesizes SeP from selenide and ATP in vitro, while SPS1 does not have this activity . Loss of function in NIH3T3 cells using RNA interference technology showed that SPS2 is required for selenoprotein biosynthesis, while SPS1 does not affect the biosynthesis of this protein class . While some insects such as the red beetle and silkworm have lost the selenoprotein synthesizing machinery including SPS2, SPS1 is still encoded in the genome of these insects, suggesting SPS1 is required for a function other than SeP synthesis .
Although SPS1 does not catalyze SeP biosynthesis, it plays essential roles in the cell. When the gene encoding SPS1 (SPS1, also designated patufet) was deleted in Drosophila, the embryo showed lethality during development , and reactive oxygen species (ROS) levels increased . The haploinsufficiency of genes involved in the Ras-regulated signaling pathway was also suppressed by SPS1 knockout in Drosophila . From the finding that the SelD (E. coli SPS) mutant of E. coli can be complemented by human SPS1 only when L-Sec is supplemented in the medium, it was suggested that SPS1 is involved in the recycling of Sec . However, the means by which SPS1 may be involved in Sec recycling has not been determined. Recently, it was found that the targeted depletion of SPS1 by RNA interference in Drosophila SL2 cells causes growth inhibition, ROS induction and megamitochondrial formation by increasing intracellular glutamine levels . Interestingly, human SPS1 was found to interact with the soluble liver antigen, which was recently identified as eukaryotic Sec synthase (SecS), and the binding reaction was enhanced by Sec tRNA methylase designated SECp43 [18, 19]. It should be noted that SecS is a pyridoxal phosphate (PLP)-dependent enzyme and, therefore, the uptake and/or activation of vitamin B6 may be related to selenium metabolism [20, 21].
Vitamin B6 is a water-soluble compound that contains a pyridine ring. Vitamin B6 is present in nature as several different forms such as pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM) and their 5'-phosphorylated forms . Before use, these vitamers are converted to PLP, which is the metabolically active form. PLP is used as a cofactor for PLP-dependent enzymes, where the pyridine ring acts as an electron sink during enzymatic reactions. Since animals, including humans, cannot synthesize vitamin B6, they must obtain it from their diet . PLP can be synthesized through several different pathways, and two types of enzymes, kinases and oxidases, participate in these pathways. For PM to be converted to PLP, it is first phosphorylated by a kinase (PL/PM/PN kinase) to form pyridoxamine phosphate (PMP), and then the PMP is oxidized to form PLP using an oxidase (PMP/PNP oxidase). PN can also be converted to PLP using the same kinase and oxidase used for PM. In this case, the phosphorylated intermediate is pyridoxine phosphate (PNP). However, PL can be directly converted to PLP by phosphorylation using a kinase . Therefore, kinases and oxidases are important enzymes for PLP synthesis.
There are more than 100 PLP-dependent enzymes in a cell that perform essential roles in various metabolic pathways including amino acid metabolism (such as amino acid synthesis and degradation), fatty acid metabolism (such as the synthesis of polyunsaturated fatty acids), and carbohydrate metabolism (such as the breakdown of glycogen)  and references therein]. The PLP-dependent enzymes that participate in amino acid metabolism can be classified into 4 categories: transaminase, racemase, decarboxylase and α,β-eliminase . Interestingly, the biosynthesis of Sec can be mediated by cystathionine β-synthase (CBS) using serine as a precursor and it can also be synthesized by cystathionine γ-lyase (CGL) from selenocystathionine [26, 27]. Both CBS and CGL are PLP-dependent enzymes . In addition, enzymes that are involved in the degradation of Sec, such as selenocysteine lyase (SCL), D-selenocystine α, and β-lyase, use PLP as a cofactor . Recently, it was found that SCL can interact with SPS1 . Therefore, it seems that vitamin B6 participates in the metabolism of Sec, i.e., in the biosynthesis and/or decomposition of Sec.
In the present study, we found that the knockdown of SPS1 led to the down regulation of genes involved in PLP biosynthesis, which, in turn, induced the formation of megamitochondria and the expression of genes responsible for innate immunity. Our findings suggest that SPS1 primarily regulates PLP biosynthesis, and the intracellular PLP level affects various biological processes such as amino acid metabolism, megamitochondrial formation and innate immune response.