Characterization of the active site and coenzyme binding pocket of the monomeric UDP- galactose 4'- epimerase of Aeromonas hydrophila

Shivani Agarwal, Neeraj Mishra, Shivangi Agarwal, Aparna Dixit

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

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 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.

Original languageEnglish (US)
Pages (from-to)419-426
Number of pages8
JournalBMB Reports
Volume43
Issue number6
DOIs
StatePublished - 2010

Keywords

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

Fingerprint

Dive into the research topics of 'Characterization of the active site and coenzyme binding pocket of the monomeric UDP- galactose 4'- epimerase of Aeromonas hydrophila'. Together they form a unique fingerprint.

Cite this