Characterization of the active site and coenzyme binding pocket of the monomeric UDP- galactose 4'- epimerase of Aeromonas hydrophila.
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.
Agarwal
Shivani
S
Jawaharlal Nehru University, New Delhi, India.
Mishra
Neeraj
N
Agarwal
Shivangi
S
Dixit
Aparna
A
D016980:Aeromonas hydrophila / Q000201:enzymology*; D055162:Biocatalysis; D002152:Calorimetry, Differential Scanning; D020134:Catalytic Domain; D005990:Glycerol / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D014534:UDPglucose 4-Epimerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D014534:UDPglucose 4-Epimerase; D005990:Glycerol