Biotechnology Letters

, Volume 31, Issue 1, pp 147–153 | Cite as

Cloning and characterization of CalS7 from Micromonospora echinospora sp. calichensis as a glucose-1-phosphate nucleotidyltransferase

  • Dinesh Simkhada
  • Tae-Jin Oh
  • Eui Min Kim
  • Jin Cheol Yoo
  • Jae Kyung SohngEmail author
Original Research Paper


The deoxysugar biosynthetic gene cluster of calicheamicin contains the calS7, which encodes glucose-1-phosphate nucleotidyltransferase and converts glucose-1-phosphate and nucleotides (NTP) to NDP-glucose and pyrophosphate. calS7 was expressed in Escherichia coli BL21(DE3), and the purified protein had significant thymidylyltransferase and uridylyltransferase activities as well, with some guanidylyltransferase activity but negligible cytidyl and adenyltransferase activity. The functions of thymidylyltransferase and uridylyltransferase were also verified using one-pot enzymatic synthesis of TMK and ACK. The products were analyzed by HPLC and ESI/MS, which showed peaks at m/z = 563 and 565 for TDP-d-glucose and UDP-d-glucose, respectively, in negative mode.


Calicheamicin Deoxysugar Micromonospora echinospora sp. calichensis Thymidylyltransferase Uridylyltransferase 



This study was supported by a grant of the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (0720260) and Korean Research Foundation (KRF) by providing research scholarship for a Ph D scholar.


  1. Ahlert J, Shepard E, Lomovskaya N et al (2002) The calicheamicin gene cluster and its iterative type I enediyne PKS. Science 297:1173–1176PubMedCrossRefGoogle Scholar
  2. Aragäo D, Marques AR, Frazäo C et al (2006) Cloning, expression, purification, crystallization and preliminary structure determination of glucose-1-phosphate uridylyltransferase (UgpG) from Sphingomonas elodea ATCC 31461 bound to glucose-1-phosphate. Acta Crystallogr Sect F Struct Biol Cryst Commun 62:930–934PubMedCrossRefGoogle Scholar
  3. Barton WA, Lesniak J, Biggins JB et al (2001) Structure, mechanism and engineering of a nucleotidylyltransferase as a first step toward glycorandomization. Nat Struct Biol 8:545–551PubMedCrossRefGoogle Scholar
  4. Biggins JB, Onwuemw KC, Thorson JS (2003) Resistance to enediyne antitumor antibiotics by CalC self-sacrifice. Science 301:1537–1541PubMedCrossRefGoogle Scholar
  5. Blankenfeldt W, Asuncion M, Lam JS et al (2000) The structural basis of the catalytic mechanism and regulation of glucose-1-phosphate thymidylyltransferase (RmlA). EMBO J 19:6652–6663PubMedCrossRefGoogle Scholar
  6. Galm U, Hager MH, Van Lanen SG et al (2005) Antitumor antibiotics: bleomycin, enediynes, and mitomycin. Chem Rev 105:739–758PubMedCrossRefGoogle Scholar
  7. Kieser T, Bibb MJ, Buttner MJ et al (2000) Practical streptomyces genetics. John Innes Foundation, NorwichGoogle Scholar
  8. Marolda CL, Valvano MA (1995) Genetic analysis of the dTDP-rhamnose biosynthesis of the E. coli Vw 187 (O7:K1) rfb gene cluster: identification of functional homologs of rfbB and rfbA in the rff cluster and correct location of the rffE gene. J Bacteriol 177:5539–5546PubMedGoogle Scholar
  9. Merson-Davies LA, Cundliffe E (1994) Analysis of five tylosin biosynthesis genes from the tylBA region of the Streptomyces fradie genome. Mol Microbiol 13:349–355PubMedCrossRefGoogle Scholar
  10. Myers AG, Cohen SB, Kwon BM (1994) DNA Cleavage by neocarzinostatin chromophore establishing the intermediacy of chromophore-derived cumulene and biradical species and their role in sequence-specific cleavage. J Am Chem Soc 116:1255–1271CrossRefGoogle Scholar
  11. Oh J, Kim BG, Sohng JK et al (2003) One-pot enzymatic production of dTDP-4-keto-6-d-glucose from dTMP and glucose-1-phosphate. Biotech Bioeng 84:452–458CrossRefGoogle Scholar
  12. Parajuli N, Lee DS, Lee HC et al (2004) Cloning, expression and characterization of glucose-1-phosphate thymidylyltransferase (strmlA) from Thermus caldophilus. Biotechnol Lett 26:437–442PubMedCrossRefGoogle Scholar
  13. Pissowotzki K, Mansouri K, Piepersberg W (1991) Genetics of streptomycin production in Streptomyces griseus: molecular structure and putative function of genes strELMB2N. Mol Gen Genet 231:113–123PubMedCrossRefGoogle Scholar
  14. Simone Z, Davide Z, Camillo R et al (2001) Kinetic and crystallographic analyses support a sequential-ordered Bi catalytic mechanism for Escherichia coli glucose-1-phosphate thymidylyltransferase. J Mol Biol 313:831–843CrossRefGoogle Scholar
  15. Sohng JK, Noh HR, Lee OH et al (2002) Function of lysine-148 in TDP-d-glucose 4, 6-dehydratase from Streptomyces antibioticus Tű99. J Microbiol Biotechnol 12:217–221CrossRefGoogle Scholar
  16. Thoden JB, Holden HM (2007a) Active site geometry of glucose-1-phosphate uridylyltransferase. Protein Sci 16:1379–1388PubMedCrossRefGoogle Scholar
  17. Thoden JB, Holden HM (2007b) The molecular architecture of glucose-1-phosphate uridylyltransferase. Protein Sci 16:432–440PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Dinesh Simkhada
    • 1
  • Tae-Jin Oh
    • 1
  • Eui Min Kim
    • 1
  • Jin Cheol Yoo
    • 2
  • Jae Kyung Sohng
    • 1
    Email author
  1. 1.Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical EngineeringSun Moon UniversityAsansi, ChungnamRepublic of Korea
  2. 2.Department of Pharmacy, College of PharmacyChosun UniversityGwangjuKorea

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