Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning and Expression of Glucose-1-Phosphate Thymidylyltransferase Gene (schS6) from Streptomyces sp. SCC-2136

  • Han, Ji-Man (Department of Biochemistry and Molecular Biology, University of Ulsan, College of Medicine) ;
  • Kim, Su-Min (Department of Pharmacy, Chosun University) ;
  • Lee, Hyo-Jung (Department of Pharmacy, Chosun University) ;
  • Yoo, Jin-Cheol (Department of Pharmacy, Chosun University)
  • Published : 2007.04.30
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Abstract

The deoxysugar biosynthetic gene cluster of Sch 47554/Sch 47555 was cloned from Streptomyces sp. SCC-2136. One of the ORFs, schS6, appeared to encode glucose-1-phosphate thymidylyltransferase, which converts dTTP and glucose-1-phosphate to TDP-D-glucose and pyrophosphate. The dTDP-D-glucose is a key metabolite in prokaryotics as a precursor for a large number of modified deoxysugars, and these deoxysugars are a maj or part of various antibiotics, ranging from glycosides to macrolides. SchS6 was expressed in E. coli vector pSCHS6 and the expressed protein was purified to apparent homogeneity by ammonium sulfate precipitation and Ni-NTA affinity column chromatography. The specific activity of the purified enzyme increased 4.7-fold with 17.5% recovery. It migrated as a single band on SDS-PAGE with an apparent molecular mass of 56kDa. The purified protein showed glucose-1-phosphate thymidylyltransferase activity, catalyzing a reversible bimolecular group transfer reaction. In the forward reaction, the highest activity was obtained with combination of dTTP and ${\alpha}-D-glucose-1-phosphate$, and only 12% of that activity was obtained with the substrates $UTP/{\alpha}-D-glucose-1-phosphate$. In the opposite direction, the purified protein was highly specific for dTDP-D-glucose and pyrophosphate.

Keywords

References

  1. Aguirrezabalaga, I., C. Olano, N. Allende, L. Rodriguez, A. F. Brana, C. Mendez, and J. A. Salas. 2000. Identification and expression of genes involved in biosynthesis of - oleandrose and its intermediate L-olivose in the oleandomycin producer Streptomyces antibioticus. Antimicrob. Agents Chemother. 44: 1266-1275 https://doi.org/10.1128/AAC.44.5.1266-1275.2000
  2. Alexander, C. W.-W. 1997. The role of carbohydrates in biologically active natural products. Nat. Prod. Rep. 14: 99-110 https://doi.org/10.1039/np9971400099
  3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  4. Choi, S. S., J. H. Kim, J.-H. Kim, D. K. Kang, S. S. Kang, and S. K. Hong. 2006. Functional analysis of sprD gene encoding Streptomyces griseus protease D (SGPD) in Streptomyces griseus. J. Microbiol. Biotechnol. 16: 312-317
  5. Chu, M., R. Yarborough, J. Schwartz, M. G. Patel, A. C. Horan, V. P. Gullo, P. R. Das, and M. S. Puar. 1992. Sch 47554 and Sch 47555, two novel antifungal antibiotics produced from a Streptomyces sp. SCC-2136. J. Antibiot. 46: 861-865
  6. Hoffmeister, D., K. Ichinose, S. Domann, B. Faust, A. Trefzer, G. Drager, A. Kirschning, C. Fischer, E. Kunzel, D. W. Bearden, J. Rohr, and A. Bechthold. 2000. The NDP-sugar co-substrate concentration and the enzyme expression level influence the substrate specificity of glycosyltransferases: Cloning and characterization of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster. Chem. Biol. 7: 821-831 https://doi.org/10.1016/S1074-5521(00)00029-6
  7. Jo, Y. Y., J. Liu, Y. Y. Jin, Y. Y. Yang, and J. W. Suh. 2005. Isolation and characterization of kasugamycin biosynthetic genes from Streptomyces kasugaensis KACC 20262. J. Microbiol. Biotechnol. 15: 491-496
  8. Kieser, T., M. J. Bibb, M. J. Buttner, K. F. Chater, and D. A. Hopwood. 2000. Practical Streptomyces Genetics. The John Innes Foundation
  9. Kil, M. W. and S. I. Chang. 1998. Expression in E. coli, purification, and characterization of the tobacco sulfonylurea herbicide-resistant recombinant acetolactate synthase and its interaction with the triazolopyrimidine herbicides. J. Biochem. Mol. Biol. 31: 287-295
  10. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680-685 https://doi.org/10.1038/227680a0
  11. Lannart, L., R. Kaiser, P. R. Reeves, and A. A. Lindberg. 1994. Purification, characterization, and high performance liquid chromatography assay of Salmonella glucose-1-phosphate cytidylyltransferase from the cloned rfb F gene. J. Biol. Chem. 269: 122-126
  12. Lee, H. C., J. K. Sohng, H. J. Kim, D. H. Nam, C. N. Seong, J. M. Han, and J. C. Yoo. 2004. Cloning, expression, and biochemical characterization of dTDP-glucose 4,6-dehydratase gene (gerE) from Streptomyces sp. GERI-155. J. Microbiol. Biotechnol. 14: 576-583
  13. Lee, H. C., J. K. Sohng, H. J. Kim, D. H. Nam, J. M. Han, S. S. Cho, J. H. Choi, and J. C. Yoo. 2004. Cloning and expression of the glucose-1-phosphate thymidylyltransferase gene (gerD) from Streptomyces sp. GERI-155. Mol. Cells 17: 274-280
  14. Lee, H. J. and H. J. Jin. 2003. Renaturation of recombinant ErmSF and its refolding behavior. Mol. Cells 16: 187-193
  15. Lindquist, L., R. Kaiser, P. R. Reeves, and A. A. Lindberg. 1993. Purification, characterization and HPLC assay of Salmonella glucose-1-phosphate thymidylyltransferase from the cloned rfbA gene. Eur. J. Biochem. 211: 763-770 https://doi.org/10.1111/j.1432-1033.1993.tb17607.x
  16. Lombo, E, K. Siems, A. E Brana, C. Mandez, K. Bindseil, and J. A. Salas. 1997. Cloning and insertional inactivation of Streptomyces argillaceus genes involved in the earliest steps of biosynthesis of the sugar moieties of the antitumor polyketide mithramycin. J. Bacteriol. 179: 3354-3357 https://doi.org/10.1128/jb.179.10.3354-3357.1997
  17. Maharjan, J., K. Liou, H. C. Lee, C. G. Kim, J. Lee, J. C. Yoo, and J. K. Sohng. 2003. Functional identification of rub52 gene involved in the biosynthesis of rubradirin. Biotech. Lett. 25: 909-915 https://doi.org/10.1023/A:1024075902143
  18. Sam brook, J. and D. W. Russel. 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, U.S.A
  19. Schein, C. H. and M. H. M. Notebom. 1988. Formation of insoluble recombinant proteins in E. coli is favoured by lower growth temperature. Bio/Technology 6: 291-294 https://doi.org/10.1038/nbt0388-291
  20. Simone, Z., Z. Davide, R. Camillo, S. Laura, T. Michela, and B. Martino. 2001. Kinetic and crystallographic analyses support a sequential-ordered Bi Bi catalytic mechanism for Escherichia coli glucose-1-phosphate thymidylyltransferase. J. Molec. Biol. 313: 831-843 https://doi.org/10.1006/jmbi.2001.5073
  21. Sohng, J. K., H. R. Noh, O. H. Lee, S. J. Kim, J. M. Han, S. K. Nam, and J. C. Yoo. 2002. Function of lysine-148 in TDP-D-glucose4,6-dehydratase from Streptomyces antibioticus Tu99. J. Microbiol. Biotechnol. 12: 217-221
  22. Walker, S., K. G. Valentine, and D. Kahne. 1990. Sugar as DNA binders: A comment on the calicheamicin oligosaccharide. J. Am. Chem. Soc. 112: 6428-6429 https://doi.org/10.1021/ja00173a058
  23. Wang, L., R. L. White, and L. C. Vining. 2002. Biosynthesis of the dideoxysugar component of jadomycin B: Genes in the jad cluster of Streptomyces venezuelae ISP5230 for L-digitoxose assembly and transfer to the angucycline aglycone. Microbiology 148: 1091-1103 https://doi.org/10.1099/00221287-148-4-1091
  24. Westrich, L., S. Domann, B. Faust, D. Bedford, D. A. Hopwood, and A. Bechthold. 1999. Cloning and characterization of a gene cluster from Streptomyces cyanogenus S136 probably involved in landomycin biosynthesis. FEMS Microbiol. Lett. 170: 381-387 https://doi.org/10.1111/j.1574-6968.1999.tb13398.x
  25. Wright, F. and M. J. Bibb. 1992. Codon usage in the G+C-rich Streptomyces genome. Gene 113: 55-65 https://doi.org/10.1016/0378-1119(92)90669-G
  26. Yang, H. Y, S. S. Choi, W. J. Chi, J. H. Kim, D. K. Kang, J. Chun, S. S. Kang, and S. K. Hong. 2005. Identification of the sprU gene encoding an additional sprT homologous trypsin-type protease in Streptomyces griseus. J. Microbiol. Biotechnol. 15: 1125-1129