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Identification of Amino-Acids Residues for Key Role in Dextransucrase Activity of Leuconostoc mesenteroides B-742CB  

Ryu, Hwa-Ja (Laboratory of Functional Carbohydrate Enzymes and Microbial Genomics, Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University)
Kim, Do-Man (Laboratory of Functional Carbohydrate Enzymes and Microbial Genomics, School of Biological Sciences and Technology, Chonnam National University)
Seo, Eun-Seong (Laboratory of Functional Carbohydrate Enzymes and Microbial Genomics, Department of Materials and Biochemical Engineering, Chonnam National University)
Kang, Hee-Kyung (Laboratory of Functional Carbohydrate Enzymes and Microbial Genomics, Engineering Research Institute, Chonnam National University)
Lee, Jin-Ha (Laboratory of Functional Carbohydrate Enzymes and Microbial Genomics, Engineering Research Institute, Chonnam National University)
Yoon, Seung-Heon (Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University)
Cho, Jae-Young (Laboratory of Functional Carbohydrate Enzymes and Microbial Genomics, Department of Materials and Biochemical Engineering, Chonnam National University)
Robyt, John-F. (Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University)
Kim, Do-Won (Department of Physics, Kangnung National University)
Chang, Suk-Sang (Ponghang Accelarator Laboratory, Pohang University of Science and Technology)
Kim, Seung-Heuk (Lifeza Co., Ltd)
Kimura, Atsuo (Graduate school of Agriculture, Kokkaido University)
Publication Information
Journal of Microbiology and Biotechnology / v.14, no.5, 2004 , pp. 1075-1080 More about this Journal
Abstract
Dextransucrase (DSRB742) from Leuconostoc mesenteroides NRRL B-742CB is a glucosyltransferase that catalyzes the synthesis of dextran using sucrose, or the synthesis of oligosaccharides when acceptor molecules, like maltose, are present. The DSRB742 gene (dsrB742) was cloned and the properties were characterized. In order to identify critical amino acid residues, the DSRB742 amino acid sequence was aligned with glucosyltransferase sequences, and three amino acid residues reported as sucrose binding amino acids in Streptococcus glucosyltransferases were selected for site-directed mutagenesis experiments. Asp-533, Asp-536, and His-643 were independently replaced with Ala or Asn. D533A and D536A dextransucrases showed reduced dextran synthesis activities, 2.3% and 40.8% of DSRB742 dextransucrase, respectively, and D533N, D536N, H643A, end H643N dextransucrases showed complete suppression of dextran synthesis activities altogether. Additionally, D536N dextransucrase showed complete suppression of oligosaccharide synthesis activities. However, modifications at Asp-533 or at His-643 retained acceptor reaction activities in the range of 8.4% to 21.3% of DSRB742 acceptor reaction activity. Thus at least two carboxyl groups of Asp-533 and Asp-536, and His-643 as a proton donor, are essential for the catalysis process.
Keywords
Dextransucrase; catalytic site; Leuconostoc mesenteroides; site-directed mutagenesis; acceptor reaction;
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