Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Role of Dipeptide at Extra Sugar-Binding Space of Thermus Maltogenic Amylase in Transglycosylation Activity

  • Baek, Jin-Sook (National Laboratory for Functional Food Carbohydrate and Center for Agricultural Bio-Materials, Seoul National University) ;
  • Kim, Tae-Jip (Department of Food Science & Technology, Chungbuk National University) ;
  • Kim, Young-Wan (National Laboratory for Functional Food Carbohydrate and Center for Agricultural Bio-Materials, Seoul National University) ;
  • Cha, Hyun-Ju (National Laboratory for Functional Food Carbohydrate and Center for Agricultural Bio-Materials, Seoul National University) ;
  • Kim, Jung-Wan (Department of Biology, University of Incheon) ;
  • Kim, Yong-Ro (National Laboratory for Functional Food Carbohydrate and Center for Agricultural Bio-Materials, Seoul National University) ;
  • Lee, Sung-Joon (Stanford School of Medicine, Palo Alto Medical Research Foundation) ;
  • Moon, Tae-Wha (National Laboratory for Functional Food Carbohydrate and Center for Agricultural Bio-Materials, Seoul National University) ;
  • Park, Kwan-Hwa (National Laboratory for Functional Food Carbohydrate and Center for Agricultural Bio-Materials, Seoul National University)
  • Published : 2003.12.01
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Abstract

Two conserved amino acid residues in the extra sugar-binding space near the catalytic site of Thermus maltogenic amylase (ThMA) were analyzed for their role in the hydrolysis and transglycosylation activity of the enzyme. Site-directed mutagenesis was carried out by replacing N33l with a lysine (N331K), E332 with a histidine (E332H), or by replacing both residues at the same time (N331K/E332H). The measured $K_m$ values indicated that affinities toward all substrates tested, including starch, pullulan, ${\beta}-cyclomaltodextrin$, and acarbose, were lower in all the mutants compared to that of wild-type ThMA, leading to reduced hydrolysis activity. In addition, the lower ratio of transglycosylation to hydrolysis in the mutants compared to that in the wild-type ThMA indicated that these mutants preferred hydrolysis to the transglycosylation reaction. These results demonstrated that the conserved dipeptide at 331 and 332 of ThMA is directly involved in the formation and accumulation of transfer products by accommodating acceptor sugar molecules.

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