Biochemical and functional characterization of UDP-galactose 4-epimerase from Aeromonas hydrophila.
Bacteria of genus Aeromonas, responsible for a variety of pathological conditions in humans and fish, are ubiquitous waterborne bacteria. Aeromonas produces several virulent factors including a complex of lipopolysaccharide and surface array protein, involved in colonization. UDP-galactose 4-epimerase (GalE) catalyzes the production of UDP-galactose, a precursor for lipopolysaccharide biosynthesis, and thus is an important drug target. GalE exhibits interspecies variation and heterogeneity at its structural and functional level and therefore, the differences between the GalE of the host and the pathogen can be exploited for drug designing. In the present study, we report biochemical and functional characterization of the recombinant GalE of Aeromonas hydrophila. Unlike GalE reported from all other species, the purified recombinant GalE of A. hydrophila was found to exist as a monomer. This is the first report of UDP-galactose 4-epimerase from any species being a monomer. The molecular mass of the 6xHis-rGalE was determined to be 38271.477 (m/z). The 6xHis-rGalE with a K(m) of 0.5 mM for UDP-galactose exhibited optimum activity at 37 degrees C and pH 8-9. Spectrofluorimetric and CD analysis confirmed that the thermal inactivation was due to structural changes and not due to the NAD-dissociation. A relatively more ordered structure of the enzyme at pH 8 and 9 as compared to that at pH 6 or 7 suggests a key role of the electrostatic interactions in maintaining its native tertiary structure.
Agarwal
Shivani
S
Gene Regulation Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India.
Gopal
Keshav
K
Upadhyaya
Tanuja
T
Dixit
Aparna
A
D016980:Aeromonas hydrophila / Q000201:enzymology*; D002384:Catalysis; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D009243:NAD / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D019998:Racemases and Epimerases / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D014534:UDPglucose 4-Epimerase / Q000737:chemistry*
D003432:Cross-Linking Reagents; D009243:NAD; D019998:Racemases and Epimerases; D014534:UDPglucose 4-Epimerase