The Drosophila selenophosphate synthetase (selD) gene is required for development and cell proliferation

To study the function of selenoproteins in development and growth we have used a lethal mutation (selD(ptuf)) of the Drosophila homologous selenophosphate synthetase (selD) gene. This enzyme is involved in the selenoprotein biosynthesis. The selD(ptuf) loss-of-function mutation causes aberrant cell proliferation and differentiation patterns in the brain and imaginal discs, as deduced from genetic mosaics, patterns of gene expression and analysis of cell cycle markers. In addition to that, selenium metabolism is also necessary for the ras/MAPKinase signal tansduction pathway. Therefore, the use of Drosophila imaginal discs and brain and in particular the selD(ptuf) mutation, provide an excellent model to investigate the role of selenoproteins in the regulation of cell proliferation, growth and differentiation.     

Selenoprotein synthesis in archaea

The availability of the genome sequences from several archaea has facilitated the identification of the encoded selenoproteins and also of most of the components of the machinery for selenocysteine biosynthesis and insertion. Until now, selenoproteins have been identified solely in species of the genera Methanococcus (M.) and Methanopyrus. Apart from selenophosphate synthetase, they include only enzymes with a function in energy metabolism. Like in bacteria and eukarya, selenocysteine insertion is directed by a UGA codon in the mRNA and involves the action of a specific tRNA and of selenophosphate as the selenium donor. Major differences to the bacterial system, however, are that no homolog for the bacterial selenocysteine synthase was found and, especially, that the SECIS element of the mRNA is positioned in the 3' nontranslated region. The characterisation of a homolog for the bacterial SelB protein showed that it does not bind to the SECIS element necessitating the activity of at least a second protein. The use of the genetic system of M. maripaludis allowed the heterologous expression of a selenoprotein gene from M. jannaschii and will facilitate the elucidation of the mechanism of the selenocysteine insertion process in the future.     

New Developments in Selenium Biochemistry: Selenocysteine Biosynthesis in Eukaryotes and Archaea

We used comparative genomics and experimental analyses to show that (1) eukaryotes and archaea, which possess the selenocysteine (Sec) protein insertion machinery contain an enzyme, O-phosphoseryl-transfer RNA (tRNA)^[Ser]Sec kinase (designated PSTK), which phosphorylates seryl-tRNA^[Ser]Sec to form O-phosphoseryl-tRNA^[Ser]Sec and (2) the Sec synthase (SecS) in mammals is a pyridoxal phosphate-containing protein previously described as the soluble liver antigen (SLA). SecS uses the product of PSTK, O-phosphoseryl-tRNA^[Ser]Sec, and selenophosphate as substrates to generate selenocysteyl-tRNA^[Ser]Sec. Sec could be synthesized on tRNA^[Ser]Sec from selenide, adenosine triphosphate (ATP), and serine using tRNA^[Ser]Sec, seryl-tRNA synthetase, PSTK, selenophosphate synthetase, and SecS. The enzyme that synthesizes monoselenophosphate is a previously identified selenoprotein, selenophosphate synthetase 2 (SPS2), whereas the previously identified mammalian selenophosphate synthetase 1 did not serve this function. Monoselenophosphate also served directly in the reaction replacing ATP, selenide, and SPS2, demonstrating that this compound was the active selenium donor. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that contain selenoproteins. X.-M. Xu and B. A. Carlson contributed equally to the studies described herein.     

Fetal mouse selenophosphate synthetase 2 (SPS2): biological activities of mutant forms in Escherichia coli.

A novel gene, sps2, detected in mouse embryo at the early stages of development has been identified as an analog of the E. coli selenophosphate synthetase gene. Unlike the E. coli enzyme, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the cDNA. Using an N-FLAG monoclonal antibody, it was shown that the full length N-FLAG-sps2 gene product was expressed in COS-7 cells. To investigate the biological activity of the sps2 gene product in vivo, the mutated sps2 gene, which contains cysteine in the place of the TGA encoded selenocysteine in the wild type, was expressed in the E. coli selD deficient mutant, MB08. Like the E. coli wild type selD gene, the mutant sps2 gene complemented the selD mutation. However, replacement of Cys with either Ala, Ser, or Thr resulted in a loss of ability to complement the selD mutation. The SPS2-CYS protein expressed in E. coli was purified and its catalytic activity was determined. The Km value for ATP was 0.75 mM and Vmax was 9.23 nmole/min/mg protein. These results confirm that the mouse embryonic sps2 gene encodes an eukaryotic selenophosphate synthetase, and that availability of selenophosphate as a selenium donor compound is widespread.     

Biosynthesis of selenophosphate

Selenophosphate synthetase, the product of the selD gene, produces the highly active selenium donor, monoselenophosphate, from selenide and ATP. Positional isotope exchange experiments have shown hydrolysis of ATP occurs by way of a phosphoryl-enzyme intermediate. Although, mutagenesis studies have demonstrated Cys17 in the Escherichia coli enzyme is essential for catalytic activity the nucleophile in catalysis has not been identified. Recently, selenophosphate synthetase enzymes have been identified from other organisms. The human enzyme which contains a threonine residue corresponding to Cys17 in the E. coli enzyme, has been overexpressed in E. coli. The purified enzyme shows no detectable activity in the in vitro selenophosphate synthetase assay. In contrast, when the human enzyme is expressed to complement a selD mutation in E. coli, in the presence of 75Se, incorporation of 75Se into bacterial selenoproteins is observed. The inactive purified human enzyme together with the very low determined specific activity of the E. coli enzyme (83 nmol/min/mg) suggest an essential component for the formation of selenophosphate has not been identified.     

Modulation of the Ras/MAPK signalling pathway by the redox function of selenoproteins in Drosophila melanogaster.

Modulation of reactive oxygen species (ROS) plays a key role in signal transduction pathways. Selenoproteins act controlling the redox balance of the cell. We have studied how the alteration of the redox balance caused by patufet (selD(ptuf)), a null mutation in the Drosophila melanogaster selenophosphate synthetase 1 (sps1) gene, which codes for the SelD enzyme of the selenoprotein biosynthesis, affects the Ras/MAPK signalling pathway. The selD(ptuf) mutation dominantly suppresses the phenotypes in the eye and the wing caused by hyperactivation of the Ras/MAPK cassette and the activated forms of the Drosophila EGF receptor (DER) and Sevenless (Sev) receptor tyrosine kinases (RTKs), which signal in the eye and wing, respectively. No dominant interaction is observed with sensitized conditions in the Wnt, Notch, Insulin-Pi3K, and DPP signalling pathways. Our current hypothesis is that selenoproteins selectively modulate the Ras/MAPK signalling pathway through their antioxidant function. This is further supported by the fact that a selenoprotein-independent increase in ROS caused by the catalase amorphic Cat(n1) allele also reduces Ras/MAPK signalling. Here, we present the first evidence for the role of intracellular redox environment in signalling pathways in Drosophila as a whole organism.     

A selenium-dependent xanthine dehydrogenase triggers biofilm proliferation in Enterococcus faecalis through oxidant production

Selenium has been shown to be present as a labile cofactor in a small class of molybdenum hydroxylase enzymes in several species of clostridia that specialize in the fermentation of purines and pyrimidines. This labile cofactor is poorly understood, yet recent bioinformatic studies have suggested that Enterococcus faecalis could serve as a model system to better understand the way in which this enzyme cofactor is built and the role of these metalloenzymes in the physiology of the organism. An mRNA that encodes a predicted selenium-dependent molybdenum hydroxylase (SDMH) has also been shown to be specifically increased during the transition from planktonic growth to biofilm growth. Based on these studies, we examined whether this organism produces an SDMH and probed whether selenoproteins may play a role in biofilm physiology. We observed a substantial increase in biofilm density upon the addition of uric acid to cells grown in a defined culture medium, but only when molybdate (Mo) and selenite (Se) were also added. We also observed a significant increase in biofilm density in cells cultured in tryptic soy broth with 1% glucose (TSBG) when selenite was added. In-frame deletion of selD, which encodes selenophosphate synthetase, also blocked biofilm formation that occurred upon addition of selenium. Moreover, mutation in the gene encoding the molybdoenzyme (xdh) prevented the induction of biofilm proliferation upon supplementation with selenium. Tungstate or auranofin addition also blocked this enhanced biofilm density, likely through inhibition of molybdenum or selenium cofactor synthesis. A large protein complex labeled with (75)Se is present in higher concentrations in biofilms than in planktonic cells, and the same complex is formed in TSBG. Xanthine dehydrogenase activity correlates with the presence of this labile selenoprotein complex and is absent in a selD or an xdh mutant. Enhanced biofilm density correlates strongly with higher levels of extracellular peroxide, which is produced upon the addition of selenite to TSBG. Peroxide levels are not increased in either the selD or the xdh mutant upon addition of selenite. Extracellular superoxide production, a phenomenon well established to be linked to clinical isolates, is abolished in both mutant strains. Taken together, these data provide evidence that an SDMH is involved in biofilm formation in Enterococcus faecalis, contributing to oxidant production either directly or alternatively through its involvement in redox-dependent processes linked to oxidant production.     

A selDABC cluster for selenocysteine incorporation in Eubacterium acidaminophilum

The four genes required for selenocysteine incorporation were isolated from the gram-positive, amino acid-fermenting anaerobe Eubacterium acidaminophilum, which expresses various selenoproteins of different functions. The sel genes were located in an unique organization on a continuous fragment of genomic DNA in the order selD1 (selenophosphate synthetase 1), selA (selenocysteine synthase), selB (selenocysteine-specific elongation factor), and selC (selenocysteine-specific tRNA). A second gene copy, encoding selenophosphate synthetase 2 (selD2), was present on a separate fragment of genomic DNA. SelD1 and SelD2 were only 62.9% identical, but the two encoding genes, selD1 and selD2, contained an in-frame UGA codon encoding selenocysteine, which corresponds to Cys-17 of Escherichia coli SelD. The function of selA, selB, and selC from E. acidaminophilum was investigated by complementation of the respective E. coli deletion mutant strains and determined as the benzyl viologen-dependent formate dehydrogenase activity in these strains after anaerobic growth in the presence of formate. selA and selC from E. acidaminophilum were functional and complemented the respective mutant strains to 83% (selA) and 57% (selC) compared to a wild-type strain harboring the same plasmid. Complementation of the E. coli selB mutant was only observed when both selB and selC from E. acidaminophilum were present. Under these conditions, the specific activity of formate dehydrogenase was 55% of that of the wild type. Transformation of this selB mutant with selB alone was not sufficient to restore formate dehydrogenase activity.     

Halving the selenophosphate synthetase gene dose confers hypersensitivity to oxidative stress in Drosophila melanogaster

Several lines of evidence indicate that selenoproteins mainly act as cellular antioxidants. Here, we test this idea comparing the sensitivity to oxidative stress (paraquat and hydrogen peroxide) between wild type and heterozygous flies for the selenophosphate synthetase selD(ptuf) mutation. Whereas under normal laboratory conditions no difference in life span is observed, a significant decrease is seen in heterozygous flies treated with oxidant agents. In contrast, overexpression of the selD gene in motoneurons did not extend longevity. Our results strongly suggest that selD haploinsufficiency makes heterozygous flies more sensitive to oxidative stress and add further evidence to the role of selenoproteins as cellular antioxidants.     

The class 2 selenophosphate synthetase gene of Drosophila contains a functional mammalian-type SECIS

Synthesis of monoselenophosphate, the selenium donor required for the synthesis of selenocysteine (Sec) is catalyzed by the enzyme selenophosphate synthetase (SPS), first described in Escherichia coli. SPS homologs were identified in archaea, mammals and Drosophila. In the latter, however, an amino acid replacement is present within the catalytic domain and lacks selenide-dependent SPS activity. We describe the identification of a novel Drosophila homolog, Dsps2. The open reading frame of Dsps2 mRNA is interrupted by an UGA stop codon. The 3'UTR contains a mammalian-like Sec insertion sequence which causes translational readthrough in both transfected Drosophila cells and transgenic embryos. Thus, like vertebrates, Drosophila contains two SPS enzymes one with and one without Sec in its catalytic domain. Our data indicate further that the selenoprotein biosynthesis machinery is conserved between mammals and fly, promoting the use of Drosophila as a genetic tool to identify components and mechanistic features of the synthesis pathway.     

Lysine-2,3-aminomutase and beta-lysine acetyltransferase genes of methanogenic archaea are salt induced and are essential for the biosynthesis of Nepsilon-acetyl-beta-lysine and growth at high salinity

The compatible solute N(epsilon)-acetyl-beta-lysine is unique to methanogenic archaea and is produced under salt stress only. However, the molecular basis for the salt-dependent regulation of N(epsilon)-acetyl-beta-lysine formation is unknown. Genes potentially encoding lysine-2,3-aminomutase (ablA) and beta-lysine acetyltransferase (ablB), which are assumed to catalyze N(epsilon)-acetyl-beta-lysine formation from alpha-lysine, were identified on the chromosomes of the methanogenic archaea Methanosarcina mazei G?1, Methanosarcina acetivorans, Methanosarcina barkeri, Methanococcus jannaschii, and Methanococcus maripaludis. The order of the two genes was identical in the five organisms, and the deduced proteins were very similar, indicating a high degree of conservation of structure and function. Northern blot analysis revealed that the two genes are organized in an operon (termed the abl operon) in M. mazei G?1. Expression of the abl operon was strictly salt dependent. The abl operon was deleted in the genetically tractable M. maripaludis. Delta(abl) mutants of M. maripaludis no longer produced N(epsilon)-acetyl-beta-lysine and were incapable of growth at high salt concentrations, indicating that the abl operon is essential for N(epsilon)-acetyl-beta-lysine synthesis. These experiments revealed the first genes involved in the biosynthesis of compatible solutes in methanogens.     

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

Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate, AMP, and orthophosphate in a 1:1:1 ratio from selenide and ATP. It was recently demonstrated that selenium delivered from selenocysteine by an E. coli NifS-like protein could replace free selenide in the in vitro SPS assay for selenophosphate formation (G. M. Lacourciere, H. Mihara, T. Kurihara, N. Esaki, and T. C. Stadtman, J. Biol. Chem. 275:23769-23773, 2000). During growth of E. coli in the presence of 0.1 microM (75)SeO(3)(2-) and increasing amounts of L-selenocysteine, a concomitant decrease in (75)Se incorporation into formate dehydrogenase H and nucleosides of bulk tRNA was observed. This is consistent with the mobilization of selenium from L-selenocysteine in vivo and its use in selenophosphate formation. The ability of E. coli to utilize selenocysteine as a selenium source for selenophosphate biosynthesis in vivo supports the participation of the NifS-like proteins in selenium metabolism.     

Selenophosphate synthetase 2 is essential for selenoprotein biosynthesis

Selenophosphate synthetase (SelD) generates the selenium donor for selenocysteine biosynthesis in eubacteria. One homologue of SelD in eukaryotes is SPS1 (selenophosphate synthetase 1) and a second one, SPS2, was identified as a selenoprotein in mammals. Earlier in vitro studies showed SPS2, but not SPS1, synthesized selenophosphate from selenide, whereas SPS1 may utilize a different substrate. The roles of these enzymes in selenoprotein synthesis in vivo remain unknown. To address their function in vivo, we knocked down SPS2 in NIH3T3 cells using small interfering RNA and found that selenoprotein biosynthesis was severely impaired, whereas knockdown of SPS1 had no effect. Transfection of SPS2 into SPS2 knockdown cells restored selenoprotein biosynthesis, but SPS1 did not, indicating that SPS1 cannot complement SPS2 function. These in vivo studies indicate that SPS2 is essential for generating the selenium donor for selenocysteine biosynthesis in mammals, whereas SPS1 probably has a more specialized, non-essential role in selenoprotein metabolism.     

Inactivation of the selB gene in Methanococcus maripaludis: effect on synthesis of selenoproteins and their sulfur-containing homologs

The genome of Methanococcus maripaludis harbors genes for at least six selenocysteine-containing proteins and also for homologs that contain a cysteine codon in the position of the UGA selenocysteine codon. To investigate the synthesis and function of both the Se and the S forms, a mutant with an inactivated selB gene was constructed and analyzed. The mutant was unable to synthesize any of the selenoproteins, thus proving that the gene product is the archaeal translation factor (aSelB) specialized for selenocysteine insertion. The wild-type form of M. maripaludis repressed the synthesis of the S forms of selenoproteins, i.e., the selenium-independent alternative system, in selenium-enriched medium, but the mutant did not. We concluded that free selenium is not involved in regulation but rather a successional compound such as selenocysteyl-tRNA or some selenoprotein. Apart from the S forms, several enzymes from the general methanogenic route were affected by selenium supplementation of the wild type or by the selB mutation. Although the growth of M. maripaludis on H(2)/CO(2) is only marginally affected by the selB lesion, the gene is indispensable for growth on formate because M. maripaludis possesses only a selenocysteine-containing formate dehydrogenase.     

腺苷甲硫氨酸合成酶的基因及结构研究进展

腺苷甲硫氨酸合成酶催化ATP和L-甲硫氨酸合成腺苷甲硫氨酸,在不同生物体和不同组织中腺苷甲硫氨酸合成酶的存在形式和编码酶的基因都有差别,本文综述了不同生物的腺苷甲硫氨酸合成酶的基因、酶结构、酶反应动力学及应用前景。     

Methanocaldococcus jannaschii prolyl-tRNA synthetase charges tRNA(Pro) with cysteine

Methanocaldococcus jannaschii prolyl-tRNA synthetase (ProRS) was previously reported to also catalyze the synthesis of cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) to make up for the absence of the canonical cysteinyl-tRNA synthetase in this organism (Stathopoulos, C., Li, T., Longman, R., Vothknecht, U. C., Becker, H., Ibba, M., and S?ll, D. (2000) Science 287, 479-482; Lipman, R. S., Sowers, K. R., and Hou, Y. M. (2000) Biochemistry 39, 7792-7798). Here we show by acid urea gel electrophoresis that pure heterologously expressed recombinant M. jannaschii ProRS misaminoacylates M. jannaschii tRNA(Pro) with cysteine. The enzyme is unable to aminoacylate purified mature M. jannaschii tRNA(Cys) with cysteine in contrast to facile aminoacylation of the same tRNA with cysteine by Methanococcus maripaludis cysteinyl-tRNA synthetase. Although M. jannaschii ProRS catalyzes the synthesis of Cys-tRNA(Pro) readily, the enzyme is unable to edit this misaminoacylated tRNA. We discuss the implications of these results on the in vivo activity of the M. jannaschii ProRS and on the nature of the enzyme involved in the synthesis of Cys-tRNA(Cys) in M. jannaschii.