BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

Characterization of the active site and coenzyme binding pocket of the monomeric UDP- galactose 4'- epimerase of Aeromonas hydrophila

  • Agarwal, Shivani (Gene Regulation Laboratory, School of Biotechnology, Jawaharlal Nehru University) ;
  • Mishra, Neeraj (Biophysical Chemistry Laboratory, School of Biotechnology, Jawaharlal Nehru University) ;
  • Agarwal, Shivangi (Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University) ;
  • Dixit, Aparna (Gene Regulation Laboratory, School of Biotechnology, Jawaharlal Nehru University)
  • Received : 2010.02.25
  • Accepted : 2010.03.04
  • Published : 2010.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

Aeromonas hydrophila is a bacterial pathogen that infects a large number of eukaryotes, including humans. The UDP-galactose 4'-epimerase (GalE) catalyzes interconversion of UDP-galactose to UDP-glucose and plays a key role in lipopolysaccharide biosynthesis. This makes it an important virulence determinant, and therefore a potential drug target. Our earlier studies revealed that unlike other GalEs, GalE of A. hydrophila exists as a monomer. This uniqueness necessitated elucidation of its structure and active site. Chemical modification of the 6xHis-rGalE demonstrated the role of histidine residue in catalysis and that it did not constitute the substrate binding pocket. Loss of the 6xHis-rGalE activity and coenzyme fluorescence with thiol modifying reagents established the role of two distinct vicinal thiols in catalysis. Chemical modification studies revealed arginine to be essential for catalysis. Site-directed mutagenesis indicated Tyr149 and Lys153 to be involved in catalysis. Use of glycerol as a cosolvent enhanced the GalE thermostability significantly.

Keywords

References

  1. Holden, H. M., Rayment, I. and Thoden, J. B. (2003) Structure and function of enzymes of the Leloir pathway for galactose metabolism. J. Biol. Chem. 278, 43885-43888. https://doi.org/10.1074/jbc.R300025200
  2. Canals, R., Jimenez, N., Vilches, S., Regue, M., Merino, S. and Tomás, J. M. (2007) Role of Gne and GalE in the Virulence of Aeromonas hydrophila Serotype O34. J. Bacteriol. 189, 540-550. https://doi.org/10.1128/JB.01260-06
  3. Gabriel, O. and Van Lanten, L. (1978) Biochemistry of Carbohydrates II; in Rev. Biochem, Manners, D. J. (ed), pp. 1-36, Bulterworths and University Park Press, London and Baltimore, MD.
  4. Mukherji, S. and Bhaduri, A. (1992) An Essential Histidine Residue for the Activity of UDP-glucose 4-Epimerase from Kluyveromyces fragilis. J. Biol. Chem. 267, 11709-11713.
  5. Mukherji, S. and Bhaduri, A. (1986) UDP-glucose 4-Epimerase from Saccharomyces fragilis: presence of an essential arginine residue at the substrate-binding site of the enzyme. J. Biol. Chem. 261, 4519-4524.
  6. Bhattacharjee, H. and Bhaduri, A. (1992) Distinct functional roles of two active site thiols in UDP-glucose 4-Epimerase from Kluyveroymces fragilis. J. Biol Chem. 267, 11714-11720.
  7. Agarwal, S., Gopal, K., Chhabra, G. and Dixit, A. (2009) Molecular cloning, sequence analysis and homology modeling of Molecular cloning, sequence analysis and homology modelling of galE encoding UDP-galactose 4-epimerase of Aeromonas hydrophila. Bioinform. 4, 216-222. https://doi.org/10.6026/97320630004216
  8. Agarwal, S., Gopal, K., Upadhyaya, T. and Dixit, A. (2007) Biochemical and functional characterization of UDP-galactose 4-epimerase from Aeromonas hydrophila. Biochim. Biophys. Acta. 1774, 828-837. https://doi.org/10.1016/j.bbapap.2007.04.007
  9. Leonard, N. J., McDonald, J. J., Hendersonn, M. E. and Reichmann, M. E. (1971) Reaction of diethyl pyrocarbonate with nucleic acid components. Biochem. 10, 3335. https://doi.org/10.1021/bi00794a003
  10. Branden, C. I., Jornvall, H., Eklund, H. and Furugren, B. (1975) The Enzymes, Boyer, P. D. (ed) 3rd, pp. 103-190, Acadmic Press, New York, USA
  11. Holbrook, J. J., Liljas, A., Steindel, S. J. and Rossmann, G. (1975) The Enzymes, Boyer, P. D. (ed) 3rd, pp. 191-292, Acadmic Press, New York, USA
  12. Harris, J. I. and Waters, M. (1976) The Enzymes, Boyer, P. D. (ed) 3rd, pp. 1-49, Acadmic Press, New York, USA.
  13. Frey, P. A. (1987) in Pyridine nucleotide coenzymes: chemical, biochemical and Medical aspects, Dolphin, D., Poulson, R. and Avramovic, O. (eds), pp. 462-511, John Wiley and Sons Inc., New York, USA.
  14. Thoden, J. B., Hegeman, A. D., Wesenberg, G., Chapeau, M. C., Frey, P. A. and Holden, H. M. (1997) Structural analysis of UDP-sugar binding to UDP-galactose 4-epimerase from Escherichia coli. Biochem. 36, 6294-6304. https://doi.org/10.1021/bi970025j
  15. Meng, F. G., Hong, Y. K., He, H. W., Lyubarev, A. E., Kurganov, B. I., Yan, Y. B. and Zhou, H. M. (2004) Osmophobic Effect of Glycerol on Irreversible Thermal Denaturation of Rabbit Creatine Kinase. Biophys. J. 87, 2247-2254. https://doi.org/10.1529/biophysj.104.044784
  16. Mathur, D. and Garg, L. C. (2007) Functional phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv: rapid purification with high yield and purity. Prot. Exp. Purif. 52, 373-378. https://doi.org/10.1016/j.pep.2006.10.006

Cited by

  1. Altered cofactor binding affects stability and activity of human UDP-galactose 4′-epimerase: Implications for type III galactosemia vol.1822, pp.10, 2012, https://doi.org/10.1016/j.bbadis.2012.05.007
  2. Novel drug target identification on UDP-Glucose 4-epimerase enzyme in Catharanthus roseus by insilico model vol.2, pp.2, 2012, https://doi.org/10.1016/S2221-1691(12)60359-1