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KMID : 0624620100430060419
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BMB Reports
2010 Volume.43 No. 6 p.419 ~ p.426
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Characterization of the active site and coenzyme binding pocket of the monomeric UDP- galactose 4’- epimerase of Aeromonas hydrophila
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Agarwal Shivani
Mishra Neeraj
Agarwal Shivangi
Dixit Aparna
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Abstract
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Aeromonas hydrophila is a bacterial pathogen that infects a large number of eukaryotes, including humans. The UDP-galactose 4’-epimerase (GalE) catalyzes interconversion of UDPgalactose 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.
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KEYWORD
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Chemical modification, Coenzyme fluorescence, Cosolvent, Site-directed mutagenesis, UDP-galactose 4’-epimerase
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