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http://dx.doi.org/10.5012/bkcs.2009.30.7.1547

Cloning and Characterization of UDP-glucose Dehydrogenase from Sphingomonas chungbukensis DJ77  

Yoon, Moon-Young (Department of Chemistry, Hanyang University)
Park, Hye-Yeon (Department of Chemistry, Hanyang University)
Park, Hae-Chul (Department of Chemistry, Hanyang University)
Park, Sung-Ha (Department of Biochemistry, Chungbuk National University)
Kim, Sung-Kun (Department of Chemistry and Biochemistry and Institute of Biomedical Studies, Baylor University)
Kim, Young-Chang (Department of Microbiology, Chungbuk National University)
Shin, Mal-shik (Department of Food and Nutrition, Chonnam National University)
Choi, Jung-Do (Department of Biochemistry, Chungbuk National University)
Publication Information
Bulletin of the Korean Chemical Society / v.30, no.7, 2009 , pp. 1547-1552 More about this Journal
Abstract
Sphingomonas chungbukensis DJ77 has the ability to produce large quantities of an extracellular polysaccharide that can be used as a gelling agent in the food and pharmaceutical industries. We identified, cloned and expressed the UDP-glucose dehydrogenase gene of S. chungbukensis DJ77, and characterized the resulting protein. The purified UDP-glucose dehydrogenase (UGDH), which catalyzes the reversible conversion of UDP-glucose to UDPglucuronic acid, formed a homodimer and the mass of the monomer was estimated to be 46 kDa. Kinetic analysis at the optimal pH of 8.5 indicated that the $K_m\;and\;V_{max}$ for UDP-glucose were 0.18 mM and 1.59 mM/min/mg, respectively. Inhibition assays showed that UDP-glucuronic acid strongly inhibits UGDH. Site-directed mutagenesis was performed on Gly9, Gly12 Thr127, Cys264, and Lys267. Substitutions of Cys264 with Ala and of Lys267 with Asp resulted in complete loss of enzymatic activity, suggesting that Cys264 and Lys267 are essential for the catalytic activity of UGDH.
Keywords
UDP-glucose dehydrogenase; Sphingomonas chungbukensis; Extracellular polysaccharide; Sitedirected mutagenesis;
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