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http://dx.doi.org/10.5483/BMBRep.2010.43.6.419

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)
Publication Information
BMB Reports / v.43, no.6, 2010 , pp. 419-426 More about this Journal
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
Chemical modification; Coenzyme fluorescence; Cosolvent; Site-directed mutagenesis; UDP-galactose 4'-epimerase;
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