Bulletin of the Korean Chemical Society

Korean Chemical Society (대한화학회)
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Cloning, Expression, and Characterization of UDP-glucose Pyrophosphorylase from Sphingomonas chungbukensis DJ77

  • Yoon, Moon-Young (Department of Chemistry, Research Institute for Natural Sciences, Hanyang University) ;
  • Lee, Kyoung-Jin (Department of Chemistry, Research Institute for Natural Sciences, Hanyang University) ;
  • Park, Hea-Chul (Department of Chemistry, Research Institute for Natural Sciences, Hanyang University) ;
  • Park, Sung-Ha (Department of Biochemistry, Chungbuk National University) ;
  • Kim, Sang-Gon (Department of Chemistry and Biochemistry and Institute of Biomedical Studies, Baylor University) ;
  • Kim, Sung-Kun (Department of Chemistry and Biochemistry and Institute of Biomedical Studies, Baylor University) ;
  • Choi, Jung-Do (Department of Biochemistry, Chungbuk National University)
  • Published : 2009.06.20
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Abstract

The bacterium Sphingomonas chungbukensis DJ77 produces the extracellular polysaccharide gellan in high yield. Gellan produced by this bacterium is widely used as a gelling agent, and the enzyme UDP-glucose pyrophosphorylase (UGP) is thought to play a key role in the gellan biosynthetic pathway. The UGP gene has been successfully cloned and over-expressed in E. coli. The expressed enzyme was purified with a molecular weight of approximately 32 kDa, as determined by a SDS-polyacrylamide gel, but the enzyme appears as ca. 63 kDa on a native gel, suggesting that the enzyme is present in a homodimer. Kinetic analysis of UDP-glucose for UGP indicates $K_m$ = 1.14 mM and $V_{max}$ = 10.09 mM/min/mg at pH 8.0, which was determined to be the optimal pH for UGP catalytic activity. Amino acid sequence alignment against other bacteria suggests that the UGP contains two conserved domains: An activator binding site and a glucose-1-phosphate binding site. Site-directed mutagenesis of Lys194, located within the glucose-1-phosphate binding site, indicates that substitution of the charge-reversible residue Asp for Lys194 dramatically impairs the UGP activity, supporting the hypothesis that Lys194 plays a critical role in the catalysis.

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