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BMB reports v.43 no.6, 2010년, pp.419 - 426   SCIE
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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  );
  • 초록

    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.


  • 주제어

    Chemical modification .   Coenzyme fluorescence .   Cosolvent .   Site-directed mutagenesis .   UDP-galactose 4'-epimerase.  

  • 참고문헌 (16)

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