Berry, MJ

Block, E

Boakye, HT

Carlson, BA

Gailer, J

George, GN

Gladyshev, VN

Glass, RS

Hatfield, DL

Jacobsen, NE

Johnson, S

Kahakachchi, C

Kaminski, R

Manley, SA

Mix, H

Pickering, IJ

Prenner, EJ

Saira, K

Skowronska, A

Tyson, JF

Uden, PC

Wu, Q

Xu, XM

Yamdagni, R

Zhang, Y

English

ScholarWorks@UMass Amherst

Insights into the chemical biology of selenium

University of Massachusetts AmherstScholarWorks@UMass AmherstChemistry Department Faculty Publication Series Chemistry2008Insights into the chemical biology of seleniumRS GlassMJ BerryE BlockHT BoakyeBA CarlsonSee next page for additional authorsFollow this and additional works at: https://scholarworks.umass.edu/chem_faculty_pubsPart of the Chemistry CommonsThis Article is brought to you for free and open access by the Chemistry at ScholarWorks@UMass Amherst. It has been accepted for inclusion inChemistry Department Faculty Publication Series by an authorized administrator of ScholarWorks@UMass Amherst. For more information, pleasecontact scholarworks@library.umass.edu.Recommended CitationGlass, RS; Berry, MJ; Block, E; Boakye, HT; Carlson, BA; Gailer, J; George, GN; Gladyshev, VN; Hatfield, DL; Jacobsen, NE;Johnson, S; Kahakachchi, C; Kaminski, R; Manley, SA; Mix, H; Pickering, IJ; Prenner, EJ; Saira, K; Skowronska, A; Tyson, JF; Uden,PC; Wu, Q; Xu, XM; Yamdagni, R; and Zhang, Y, "Insights into the chemical biology of selenium" (2008). Phosphorus Sulfur andSilicon and the Related Elements. 999.Retrieved from https://scholarworks.umass.edu/chem_faculty_pubs/999AuthorsRS Glass, MJ Berry, E Block, HT Boakye, BA Carlson, J Gailer, GN George, VN Gladyshev, DL Hatfield, NEJacobsen, S Johnson, C Kahakachchi, R Kaminski, SA Manley, H Mix, IJ Pickering, EJ Prenner, K Saira, ASkowronska, JF Tyson, PC Uden, Q Wu, XM Xu, R Yamdagni, and Y ZhangThis article is available at ScholarWorks@UMass Amherst: https://scholarworks.umass.edu/chem_faculty_pubs/999Insights into the Chemical Biology of SeleniumRichard S. Glass,1 Marla J. Berry,2 Eric Block,3Harriet T. Boakye,4 Bradley A. Carlson,5 Ju¨rgen Gailer,6Graham N. George,7 Vadim N. Gladyshev,8Dolph L. Hatfield,9 Neil E. Jacobsen,1 Sherida Johnson,3Chethaka Kahakachchi,4 Rafal Kamin´ski,9Shawn A. Manley,6 Heiko Mix,8 Ingrid J. Pickering,7Elmar J. Prenner,10 Kazima Saira,8Aleksandra Skowron´ska,9 Julian F. Tyson,4Peter C. Uden,4 Qiao Wu,6 Xue-Ming Xu,5Raghav Yamdagni,6 and Yan Zhang81Department of Chemistry, The University of Arizona, Tucson,AZ, USA2Department of Cell and Molecular Biology, University of Hawaii atManoa, Honolulu, HI, USA3Department of Chemistry, University at Albany, SUNY, Albany,NY, USA4Department of Chemistry, University of Massachusetts, Amherst,MA, USA5Laboratory of Cancer Prevention, Center for Cancer Research,National Cancer Institute, Bethesda, MD, USA6Department of Chemistry, University of Calgary, Calgary,Alberta, Canada7Department of Geological Sciences, University of Saskatchewan,Saskatoon, Saskatchewan, Canada8Department of Biochemistry, University of Nebraska, Lincoln,NE, USA9Center of Molecular and Macromolecular Sciences, Polish Academy ofSciences, Lodz, Poland10Department of Biological Sciences, University of Calgary, Calgary,Alberta, CanadaThe long-sought pathway by which selenocysteyl-tRNA[Ser]Sec is synthesized ineukaryotes has been revealed. Seryl-tRNA[Ser]Sec is O-phosphorylated and SecS,The support of this research by the U.S. National Science Foundation, the U.S. Na-tional Institutes of Health, and the Petroleum Research Fund, administered by the Amer-ican Chemical Society, is gratefully acknowledged.Address correspondence to Richard S. Glass, Department of Chemistry, The Univer-sity of Arizona, Tucson, AZ 85721, USA. E-mail: rglass@arizona.edua pyridoxal phosphate-dependent protein, catalyzes the reaction of O-phosphoseryl-tRNA[Ser]Sec with monoselenophosphate to give selenocysteyl-tRNA[Ser]Sec. 1H-{77Se} HMQC-TOCSY NMR spectroscopy has been developed to detect theselenium-containing amino acids present in selenized yeast after protease XIV di-gestion. An archived selenized yeast sample is found to contain the novel aminoacid S-(methylseleno)cysteine in addition to selenomethionine. Arsenite and selen-ite react with GSH to form (GS)2AsSe−. The structure of this compound has beendetermined by EXAFS, 77Se NMR and Raman spectroscopic and chromatographicstudies. Its formation under biological conditions has been demonstrated.INTRODUCTIONSelenium plays an important role in biology. It has long been recognizedas an essential mineral. Selenocysteine is the twenty-first amino acidincorporated cotranslationally into proteins.1 Selenium administeredin 200 μg doses per day as selenized yeast has been shown to decreasethe onset of colon, prostate and lung cancer in humans by as much as50%.2 However, selenium also shows a dark side. It is toxic and themargin of safety is relatively modest. This paper presents vignettesrelevant to selenium’s role in each of these areas in which chemicalstudies contributed significantly to revealing the role of selenium.Selenoproteins occur widely in archaea, prokaryotes andeukaryotes.3 A number of them have been isolated and charac-terized. Typically the selenium occurs as selenocysteine in theseproteins. It was discovered that in the genes for two of these proteins;glutathione peroxidase4 and formate dehydrogenase,5 the codon UGAwas found in the position corresponding to that for selenocysteinein the protein. Since UGA is a well-known stop codon, how does thebiological machinery know when to incorporate selenocysteine andwhen to terminate protein synthesis? It was discovered that threeadditional factors were required for selenocysteine incorporation. Aselenocysteine insertion sequence (SECIS) in the mRNA is needed. Inprokaryotes this is a stem-loop structure next to the UGA codon onthe 3′-side. In eukaryotes the SECIS is in the 3′-untranslated regionof selenoprotein mRNA. A special elongation factor is also required.Finally, a special tRNA charged with selenocysteine is essential.An unusual quaternary complex forms at the ribosome ensuringselenocysteine incorporation.6The selenocysteyl-tRNA is unusual not only for its tRNA structure,but also because it is the only known tRNA in eukaryotes that is notaminoacylated directly with the amino acid which it delivers to the ri-bosome. Rather, it is aminoacylated with serine which is then convertedto selenocysteine while on the tRNA. In prokaryotes the attached ser-ine is dehydrated by the pyridoxal phosphate-dependent Sec synthaseto afford the dehydroalanyl moiety. A selenium donor then converts thedehydroalanyl moiety into selenocysteyl-tRNA.RESULTS AND DISCUSSIONThe selenium donor which converts dehydroalanyl-tRNA intoselenocysteyl-tRNA is monoselenophosphate 1. This species is biosyn-thesized in a reaction catalyzed by seleno-HSe− + ATP −→ SePO3H3−xx + AMP + OPO3H3−Xx1(1)phosphate synthase as shown in Equation (1). The identity ofmonoselenophosphate was established with certainty by comparisonwith the authentic chemical compound.7 Monoselenophosphate waschemically synthesized in the following way. Reaction of the knownO,O,O-trimethylselenophosphate 2 with trimethylsilyl iodide gaveO,O,O-tris(trimethylsilyl)selenophosphate 3 as shown in Equation(2). Although 3 hydrolyzes in water to(MeO)3PSe + 3TMSI −→ (TMSO)3PSe + 3Mel2 3(2)give monoselenophosphate, this procedure is inconvenient because 3is not water soluble. A more preferable procedure is first to cleave onetrimethylsilyl group as shown in Equation (3) to givewater-soluble salt 4. Addition of 4 to aqueous buffers affords monose-lenophosphate 1 which must be rigorously protected from air since itis easily decomposed on oxidation [8]. Monoselenophosphate 1 couldbe characterized spectroscopically and by elemental analysis of its pre-cipitated barium salt. As expected, salt 4 selectively methylates on se-lenium, not oxygen, on treatment with methyl iodide. Further chem-ical reactions of monoseleno-phosphate were studied as well.9 Chem-ically prepared monoselenophosphate and the enzymatically synthe-sized selenium donor compound were shown to be identical by 31P NMRspectroscopy, chromatographic behavior and biological activity. Monose-lenophosphate was also shown to be the source of the selenium in themetalloid modified base (2-seleno-5-methylaminouracil) that occurs insome tRNAs found in anaerobic bacteria.10Although this biosynthesis of selenocysteyl-tRNA in prokaryotes wasworked out many years ago, that in eukaryotes and archaea was uncov-ered only very recently.11,12 The enzymes that catalyzed the conversionof the serine attached to the tRNA remained elusive. A comparative ge-nomics analysis revealed genes that co-occur with selenoproteins. Thehydroxyl group of seryl-tRNA[Ser]Sec is first phosphorylated to give O-phosphoseryl-tRNA[Ser]Sec in a reaction catalyzed by O-phosphoseryl-tRNA[Ser]Sec kinase. The selenium donor is biosynthesized in a reactioncatalyzed by the enzyme SPS2. Then the enzyme SecS catalyzes the re-action of O-phosphoseryl-tRNA[Ser]Sec and the selenium donor to formselenocysteyl-tRNA[Ser]Sec. SecS is a pyridoxal phosphate-dependentprotein that was previously identified in mammals as SLA (soluble liverantigen). That the selenium donor is monoselenophosphate was shownby the formation of Sec from phosphoseryl-tRNA[Ser]Sec, chemically gen-erated monoselenophosphate and mouse SecS.11As pointed out in the Introduction, daily administration of selenizedyeast to humans dramatically reduced the onset of three importanttypes of cancer. Identification of the chemical forms of selenium (sele-nium speciation) in biological material is a challenging task becauseof the complexity of the system. Typically the material is enzymati-cally hydrolyzed. The complex aqueous solution thus obtained is sep-arated into its components by HPLC or GC after derivatization. Thecomponents are analyzed by selenium specific ICP-MS (inductively cou-pled plasma-mass spectrometry) or GC-AED-MS (gas chromatography-atomic emission detection-mass spectrometry). As an alternative tothese established methodologies we applied 77Se NMR spectroscopicanalysis. The advantage to this method is that it could be applied di-rectly to the complex aqueous solution after enzymatic hydrolysis with-out the need for separation. Furthermore, NMR spectroscopic data arerich in information about chemical structure. Thus deduction of chem-ical structures of unknown compounds by NMR spectroscopic methodsis feasible.Selenium-77 is an attractive nucleus for NMR spectroscopy for sev-eral reasons. It has spin = 1/2 and occurs in 7.6% natural isotopic abun-dance. Although it is not as sensitive as 1H, its sensitivity is compara-ble to 13C. Furthermore, it has a chemical shift range of 3000 ppm.13Measuring 77Se by inverse 1H detection using 1H-{77Se}HMQC spec-troscopy is advantageous because this results in an increase in sensitiv-ity by a factor of almost 70.14 A further advantage to this methodology isthat only those 1H nuclei spin-spin coupled to 77Se are detected. Conse-quently, in a complex mixture of 77Se-containing compounds and thosenot containing 77Se, only the 77Se-containing compounds are detected.This results in a dramatic simplification of the 1H NMR spectrum ofthe complex mixture obtained on proteolysis of the selenized yeast. The2D 1H-{77Se}HMQC spectrum obtained with a protease XIV digestionof a recent selenized yeast tablet prepared for use in a current coloncancer study showed both 1H and 77Se chemical shifts correspondingto selenomethionine. Similar studies on an archived sample from theClark studies showed the presence of a selenium compound in additionto selenomethionine. To ascertain the structure of this additional com-ponent, more NMR information was required. Since only the protons J-coupled to 77Se are observed in these spectra and their 1D counterparts,a TOCSY sequence was added.15 With authentic selenomethionine, sig-nals were now observed for not only the Me and γ -hydrogens but α- andβ-, as well. This methodology was also applied to synthetic selenoaminoacid derivatives 5a and 6a that might be related to the unidentified se-leno compound in the selenized yeast extracts. Interestingly, only theMe signal for 5a wasMeSeSCH2CH(NHR1)CO2R5a, R = Et, R1= CO2Etb, R = R1= HMeSSeSCH2CH(NHR1)CO2R6a, R = Et, R1= CO2Etb, R = R1= Hdetected. This could mean that the coupling constant between 77Se andthe β-hydrogens through sulfur is very small. However, 6a showed α,β and Me hydrogen signals. Indeed the coupling of 77Se with the Mehydrogens through sulfur in this case is about 4.7 Hz. Consequently,further optimization of the spectrum of 5a is expected to reveal thesignals for the α, β and γ -hydrogens. Analysis of the spectra of theunknown component in the hydrolysate obtained from the Clark samplerevealed that it is S-(methylseleno)cysteine 5b and not the isomeric 6b.It has already been mentioned that selenium is toxic. It is also well-known that arsenic is toxic as well. Indeed this is evidenced by the ar-senocosis found in Bangladesh due to the presence of inorganic arsenicin the public water supply. Remarkably, mixtures of selenite and arsen-ite simultaneously administered to mammals ameliorate each others’toxicity. Furthermore, this effect is greatest with a 1:1 ratio of Se:Aswhich suggests the in vivo formation of a stoichiometric compound. Toexplore this possibility chemically, glutathione (GSH), which is the mostprevalent reducing agent in cells, was mixed in varying ratios with se-lenite and arsenite. When the ratio was that shown in Equation (4), anew compound 7 was formed in which there is a 1:1 ratio of Se:As. Thestructure of this compound was elucidated by8GSH + As(OH)3 + SeO3H− −→ (GS)2AsSe− + 3GSSG + 6H2O7(4)EXAFS, 77Se NMR, Raman spectroscopic and chromatographicstudies.16 Although this compound is formed chemically, is it formedbiologically? Indeed the bile from rabbits injected with selenite and ar-senite is found by EXAFS studies to contain substantial amounts of 7.Furthermore, 7 is assembled in erythrocyte lysate.17 The mechanismfor formation of 7 has been studied.18 An exciting application of thebeneficial effect of selenium on arsenic toxicity is the use of dietaryselenium supplements, or other forms of selenium, to counter arsenictoxicity.19In summary, the use of chemical synthesis, spectroscopic methodsand chemical insight contributed significantly to our present under-standing of the role of selenium in biology. Achievement of the resultspresented above required the collaboration of a diverse group of scien-tists in the U.S., Canada and Poland.REFERENCES[1] D. L. Hatfield, B. A. Carlson, X. -M. Xu, H. Mix, and V. N. Gladyshev, Prog. NucleicAcid Res. Mol. Biol., 81, 97 (2006).[2] L. C. Clark, G. F. Combs, Jr., B. W. Turnbull, E. H. Slate, D. K. Chalker, J. Chow, L.S. Davis, R. A. Glover, G. F. Graham, E. G. Gross, A. Kongract, J. L. Lesher, H. K.Vark, B. B. Sanders, Jr., C. L. Smith, and J. R. Taylor, J. Am. Med. Assoc., 276, 1957(1996).[3] M. Birringer, S. Pilawa, and L. Flohe´, Nat. Prod. Rep., 19, 693 (2002).[4] I. Chambers, J. Frampton, P. Goldfarb, N. Affara, W. McBain, and P.R. Harrison,EMBO J., 5, 1221 (1986).[5] F. Zinoni, A. Birkmann, T. C. Stadtman, and A. Bo¨ck, Proc. Natl. Acad. Sci. U.S.A.,83, 4560 (1986).[6] D. M. Driscoll and P. R. Copeland, Annu. Rev. Nutr., 23, 17 (2003).[7] R. S. Glass, W. P. Singh, W. Jung, Z. Veres, T. D. Scholz, and T. C. Stadtman, Bio-chemistry, 32, 12555 (1993).[8] T. C. Stadtman and R. S. Glass in Methods in Enzymology, L. Packer, Ed. (Academic,Orlando, FL, 1995), pp. 252, 309.[9] R. KamiÒski, R. S. Glass, T. B. Schroeder, J. Michalski, and A. SkowroÒska, Bioorg.Chem., 24, 247 (1997).[10] Z. Veres, L. Tsai, T. D. Scholz, M. Politino, R. S. Balaban, and T. C. Stadtman, Proc.Natl. Acad. Sci. U.S.A., 89, 2975 (1992).[11] X. -M. Xu, B. A. Carlson, H. Mix, Y. Zhang, K. Saira, R. S. Glass, M. J. Berry, V. N.Gladyshev, and D. L. Hatfield, PLoS Biol., 5, 96 (2007).[12] J. Yuan, S. Palioura, J. C. Salazar, D. Su, P. O’Donoghue, M. J. Hohn, A. M. Cardoso,W. B. Whitman, and D. So¨ll, Proc. Natl. Acad. Sci. U.S.A., 103, 18, 923 (2006).[13] H. Duddeck, Prog. Nucl. Magn. Reson. Spectrosc., 27, 1 (1995).[14] T.B. Schroeder, C. Job, M.F. Brown, and R.S. Glass, Magn. Reson. Chem., 33, 191(1995).[15] E. Block, R.S. Glass, N.E. Jacobsen, S. Johnson, C. Kahakachchi, R. KamiÒski, A.Skowron´ska, H.T. Boakye, J.F. Tyson and P.C. Uden, J. Agric. Food Chem., 52, 3761(2004).[16] J. Gailer, G. N. George, I. J. Pickering, R. C. Prince, S. C. Ringwald, J. E. Pemberton,R. S. Glass, M. S. Younis, D. W. DeYoung, and H. V. Aposhian, J. Am. Chem. Soc.,122, 4637 (2000).[17] S. A. Manley, G. N. George, I. J. Pickering, R. S. Glass, E. J. Prenner, R. Yamdagni,Q. Wu, and J. Gailer, Chem. Res. Toxicol., 19, 601 (2006).[18] J. Gailer, G. N. George, I. J. Pickering, G .A. Buttigieg, M. B. Denton, and R. S. Glass,J. Organomet. Chem., 650, 108 (2002).[19] (a) http://www.bangladesh-selenium.org; (b) S. A. Mousa, L. O’Connor, T. G. Ross-man, and E. Block, Carcinogenesis, 28, 962 (2007).

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Insights into the chemical biology of selenium

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