Binding Stoichiometry of a Recombinant Selenophosphate Synthetase with One Synonymic Substitution E197D to a Fluorescent Nucleotide Analog of ATP, TNP-ATP
The transformation of the strain - with plasmid vector pET11a containing the cloned gene of bacterial selenophosphate synthetase (SPS), selD, from the E. coli BL21-Gold (DE3) strain gives an overproducing strain of SPS with one synonymic substitution, E197D. The transformation efficiency was estimated as 8 × 108 CFU/μg plasmid DNA. 28？mg of highly purified preparation of recombinant SPS capable of binding TNP-ATP was eluted from DEAE-Sephadex column in amount of 15 % from the total soluble protein in crude extract. The fluorescent derivative of ATP, 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine-5′-triphosphate (TNP-ATP), was used as a synthetic analog of the substrate for the monitoring and quantitative analysis of the functional activity of SPS. The non-linear regression analysis of the saturation curve of TNP-ATP binding to D197 SPS with GraphPad Prism software fits to a model with 2 distinct binding sites with different in order. The SPS existence in a form of tetramer in given reaction conditions, in accordance with the concentration stoichiometry of 4 moles of TNP-ATP to 1 mole of recombinant protein, is being discussed. The tetramer structure was predicted with molecular modelling software YASARA and modelled in vacuum using steepest descent minimization energy method. We hypothesize here the recombinant SPS exists as a dimer in solution with two active sites capable of ATP binding in each subunit. 1. Introduction Selenium may be incorporated into a protein body as an aminoacid selenocysteine. Selenophosphate synthetase (EC 2.7.9.3.), a product of selD gene in bacteria, catalyzes selenophosphate formation with the help of ATP and Mg2+ ions [1]. This reaction appears common for all living organisms and serves to transform selenium into biologically active form—selenophosphate. Then, in bacteria selenoprotein synthesis is performed during a few definite steps. First, selenocysteine-specific tRNA ( ) is aminoacylated with serine—and this tRNA is unique for this aminoacid! It means that selenocysteine has its own tRNA—it is a aminoacid in the genetic code. After that, the formated product is easily converted to selenocysteine— with selenophosphate made at the previous step [2]. Further, selenocysteine formed could be incorporated into protein molecule that is encoded with UGA codon [3]. Thus, in contrary to the other aminoacids, the selenocysteine biosynthesis is carried out on its tRNA [4, 5]. In eubacteria, monoselenophosphate could be used as a selenium donor as for incorporation of selenium into the protein body as far as for conversion of thiouridine
References
[1]
G. M. Lacourciere, R. L. Levine, and T. C. Stadtman, “Direct detection of potential selenium delivery proteins by using an Escherichia coli strain unable to incorporate selenium from selenite into proteins,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 14, pp. 9150–9153, 2002.
[2]
X. M. Xu, B. A. Carlson, R. Irons et al., “Selenophosphate synthetase 2 is essential for selenoprotein biosynthesis,” Biochemical Journal, vol. 404, no. 1, pp. 115–120, 2007.
[3]
T. Tamura, S. Yamamoto, M. Takahata et al., “Selenophosphate synthetase genes from lung adenocarcinoma cells: Sps1 for recycling L-selenocysteine and Sps2 for selenite assimilation,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 46, pp. 16162–16167, 2004.
[4]
W. Leinfelder, T. C. Stadtman, and A. B？ck, “Occurrence in vivo of selenocysteyl-tRNA (SERUCA) in Escherichia coli: effect of sel mutations,” Journal of Biological Chemistry, vol. 264, pp. 9720–9723, 1989.
[5]
B. J. Lee, P. J. Worland, J. N. Davis, T. C. Stadtman, and D. L. Hatfield, “Identification of a selenocysteyl-tRNA(Ser) in mammalian cells that recognizes the nonsense codon, UGA,” Journal of Biological Chemistry, vol. 264, no. 17, pp. 9724–9727, 1989.
[6]
J. Yuan, S. Palioura, J. C. Salazar, et al., “RNA-dependent conversion of phosphoserine forms selenocysteine in eukaryotes and archaea,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, pp. 18923–18927, 2006.
[7]
S. C. Low, J. W. Harney, and M. J. Berry, “Cloning and functional characterization of human selenophosphate synthetase, an essential component of selenoprotein synthesis,” Journal of Biological Chemistry, vol. 270, no. 37, pp. 21659–21664, 1995.
[8]
E. V. Kudryashova, A. K. Gladilin, and A. V. Levashova, “Proteins in super-molecule essembly: investigation of the structure with the method of time-allowed fluorescent anysotropy,” Advances of Biological Chemistry, vol. 44, pp. 257–294, 2002 (Russian).
[9]
“pET system vectors and hosts,” Instruction manual, http://www.genomics.agilent.com/files/Manual/211521.pdf.
[10]
J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning Laboratory Manual, Cold Spring Harbor Laboratory, New York, NY, USA, 1998.
[11]
M. Osborn and C. Smith, Molecular Microbial Ecology, Taylor & Francis, New York, NY, USA, 2005.
[12]
J. Sambrook and D. W. Russel, The Condenced Protocols: From Molecular Cloning, Cold Spring Harbor Laboratory, New York, NY, USA, 2006.
[13]
Mode of access: electronic resource, 2009, http://www.invitrogen.com.
[14]
Z. Veres, I. Y. Kim, T. D. Scholz, and T. C. Stadtman, “Selenophosphate synthetase: enzyme properties and catalytic reaction,” Journal of Biological Chemistry, vol. 269, no. 14, pp. 10597–10603, 1994.
[15]
Y. Preabrazhenskaya and T. Stadtman, “Stoichiometric relations between ATP- and its fluorescent derivative TNP-ATP-binding to selenophosphate synthetase from E.coli,” in Proceedings of the 4th Annual NHLBI DIR Fellows Retreat, p. 24, Bethesda, MD, USA, 2006.
[16]
Y. V. Preobrazhenskaya, N. A. Kazak, K. A. Mandrik, and A. A. Maskevich, “Viability of E.coli after transformation with the vector pET11a, containing the inserted gene of selenophosphate synthetase,” Ecological Bulletin, vol. 1, no. 15, pp. 21–27, 2011 (Russian).
[17]
M. D. Wolfe, “Mechanistic Insights Revealed Through Characterization of a Novel Chromophore in Selenophosphate Synthetase from Escherichia coli,” IUBMB Life, vol. 55, no. 12, pp. 689–693, 2003.
[18]
D. Krepkiy and H. M. Miziorko, “Identification of active site residues in mevalonate diphosphate decarboxylase: implications for a family of phosphotransferases,” Protein Science, vol. 13, no. 7, pp. 1875–1881, 2004.
[19]
S. D. Warfolomeev and K. G. Gurevich, Biokinetics: Practical Course, Fair Press, 1999.
[20]
D. W. Martin and J. R. Sachs, “Ligands presumed to label high affinity and low affinity ATP binding sites do not interact in an (αβ)2 diprotomer in duck nasal gland Na+,K+-ATPase, nor do the sites coexist in native enzyme,” Journal of Biological Chemistry, vol. 275, no. 32, pp. 24512–24517, 2000.
[21]
E. Matsumoto, S. I. Sekine, R. Akasaka et al., “Structure of an N-terminally truncated selenophosphate synthetase from Aquifex aeolicus,” Acta Crystallographica, vol. 64, no. 6, pp. 453–458, 2008.
[22]
P. Daniluk and B. Lesyng, “A novel method to compare protein structures using local descriptors,” BMC Bioinformatics, vol. 12, p. 344, 2011.
[23]
M. Hirosawa-Takamori, H. J？ckle, and G. Vorbrüggen, “The class 2 selenophosphate synthetase gene of Drosophila contains a functional mammalian-type SECIS,” EMBO Reports, vol. 1, no. 5, pp. 441–446, 2000.
[24]
I. Y. Kim and T. C. Stadtman, “Effects of monovalent cations and divalent metal ions on Escherichia coli selenophosphate synthetase,” Proceedings of the National Academy of Sciences of the United States of America, vol. 91, no. 15, pp. 7326–7329, 1994.
[25]
I. Y. Kim, Z. Veres, and T. C. Stadtman, “Escherichia coli mutant SELD enzymes,” Journal of Biological Chemistry, vol. 267, pp. 19650–19654, 1992.
[26]
R. C. Stewart, R. VanBruggen, D. D. Ellefson, and A. J. Wolfe, “TNP-ATP and TNP-ADP as probes of the nucleotide binding site of cheA, the histidine protein kinase in the chemotaxis signal transduction pathway of Escherichia coli,” Biochemistry, vol. 37, no. 35, pp. 12269–12279, 1998.
