Bulletin of the Korean Chemical Society

Korean Chemical Society (대한화학회)
권호 표지
DOI QR코드

DOI QR Code

Cloning and Characterization of UDP-glucose Dehydrogenase from Sphingomonas chungbukensis DJ77

  • Yoon, Moon-Young (Department of Chemistry, Hanyang University) ;
  • Park, Hye-Yeon (Department of Chemistry, Hanyang University) ;
  • Park, Hae-Chul (Department of Chemistry, Hanyang University) ;
  • Park, Sung-Ha (Department of Biochemistry, Chungbuk National University) ;
  • Kim, Sung-Kun (Department of Chemistry and Biochemistry and Institute of Biomedical Studies, Baylor University) ;
  • Kim, Young-Chang (Department of Microbiology, Chungbuk National University) ;
  • Shin, Mal-shik (Department of Food and Nutrition, Chonnam National University) ;
  • Choi, Jung-Do (Department of Biochemistry, Chungbuk National University)
  • Published : 2009.07.20
Download PDF
⟨ Previous Next ⟩

Abstract

Sphingomonas chungbukensis DJ77 has the ability to produce large quantities of an extracellular polysaccharide that can be used as a gelling agent in the food and pharmaceutical industries. We identified, cloned and expressed the UDP-glucose dehydrogenase gene of S. chungbukensis DJ77, and characterized the resulting protein. The purified UDP-glucose dehydrogenase (UGDH), which catalyzes the reversible conversion of UDP-glucose to UDPglucuronic acid, formed a homodimer and the mass of the monomer was estimated to be 46 kDa. Kinetic analysis at the optimal pH of 8.5 indicated that the $K_m\;and\;V_{max}$ for UDP-glucose were 0.18 mM and 1.59 mM/min/mg, respectively. Inhibition assays showed that UDP-glucuronic acid strongly inhibits UGDH. Site-directed mutagenesis was performed on Gly9, Gly12 Thr127, Cys264, and Lys267. Substitutions of Cys264 with Ala and of Lys267 with Asp resulted in complete loss of enzymatic activity, suggesting that Cys264 and Lys267 are essential for the catalytic activity of UGDH.

Keywords

References

  1. Kim, S. J.; Chun, J.; Bae, K. S.; Kim, Y. C. Int. J. Syst. Evol. Microbiol. 2000, 50, 1641 https://doi.org/10.1099/00207713-50-4-1641
  2. Sutherland, I. W. Trends Biotechnol. 1998, 16, 41 https://doi.org/10.1016/S0167-7799(97)01139-6
  3. Kuo, C. C.; Chen, H. H.; Wang, S. P.; Grayston, J. T. J. Clin. Microbiol. 1986, 24, 1034
  4. Martins, L. O.; Sa-Correia, I. Biotechnol. Appl. Biochem. 1991, 14, 357
  5. Sa-Correia, I.; Fialho, A. M.; Videira, P.; Moreira, L. M.; Marques, A. R.; Albano, H. J. Ind. Microbiol. Biotechnol. 2002, 29, 170 https://doi.org/10.1038/sj.jim.7000266
  6. Marques, A. R.; Ferreira, P. B.; Sa-Correia, I.; Fialho, A. M. Mol. Genet. Genomics 2003, 268, 816
  7. Axelrod, J.; Kalckar, H. M.; Maxwell, E. S.; Strominger, J. L. J. Biol. Chem. 1957, 224, 79
  8. Nelsestuen, G. L.; Kirkwood, S. J. Biol. Chem. 1971, 246, 3824
  9. Ridley, W. P.; Houchins, J. P.; Kirkwood, S. J. Biol. Chem. 1975, 250, 8761
  10. Ordman, A. B.; Kirkwood, S. Biochim. Biophys. Acta 1977, 481, 25 https://doi.org/10.1016/0005-2744(77)90133-4
  11. Ordman, A. B.; Kirkwood, S. J. Biol. Chem. 1977, 252, 1320
  12. Ge, X.; Campbell, R. E.; van de Rijn, I.; Tanner, M. E. J. Am. Chem. Soc. 1998, 120
  13. Campbell, R. E.; Tanner, M. E. J. Org. Chem. 1999, 64, 9487 https://doi.org/10.1021/jo991092h
  14. Campbell, R. E.; Mosimann, S. C.; van De Rijn, I.; Tanner, M. E.; Strynadka, N. C. Biochemistry 2000, 39, 7012 https://doi.org/10.1021/bi000181h
  15. Kim, U. J.; Shizuya, H.; de Jong, P. J.; Birren, B.; Simon, M. I. Nucleic Acids Res. 1992, 20, 1083 https://doi.org/10.1093/nar/20.5.1083
  16. Kim, C. G.; Fujiyama, A.; Saitou, N. Genomics 2003, 82, 571 https://doi.org/10.1016/S0888-7543(03)00174-5
  17. Laemmli, U. K. Nature 1970, 227, 680 https://doi.org/10.1038/227680a0
  18. Bradford, M. M. Anal. Biochem. 1976, 72, 248 https://doi.org/10.1016/0003-2697(76)90527-3
  19. Ke, S. H.; Madison, E. L. Nucleic Acids Res. 1997, 25, 3371 https://doi.org/10.1093/nar/25.16.3371
  20. Sommer, B. J.; Barycki, J. J.; Simpson, M. A. J. Biol. Chem. 2004, 279, 23590 https://doi.org/10.1074/jbc.M401928200
  21. Wegrowski, Y.; Perreau, C.; Bontemps, Y.; Maquart, F. X. Biochem. Biophys. Res. Commun. 1998, 250, 206 https://doi.org/10.1006/bbrc.1998.9262
  22. Kleiger, G.; Eisenberg, D. J. Mol. Biol. 2002, 323, 69 https://doi.org/10.1016/S0022-2836(02)00885-9

Cited by

  1. Cloning and characterization of phosphomannose isomerase from sphingomonas chungbukensis DJ77 vol.42, pp.8, 2009, https://doi.org/10.5483/bmbrep.2009.42.8.523