title: Identification of Catalytic Amino Acid Residues by Chemical Modification in Dextranase.
abstract: A novel endodextranase isolated from Paenibacillus sp. was found to produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides with a degree of polymerization of 7-14 from dextran. To determine the active site, the enzyme was modified with 1-ethyl-3-[3- (dimethylamino)-propyl]-carbodiimide (EDC) and α-epoxyalkyl α-glucosides (EAGs), an affinity labeling reagent. The inactivation followed pseudo first-order kinetics. Kinetic analysis and chemical modification using EDC and EAGs indicated that carboxyl groups are essential for the enzymatic activity. Three Asp and one Glu residues were identified as candidate catalytic amino acids, since these residues are completely conserved across the GH family of 66 enzymes. Replacement of Asp189, Asp340, or Glu412 completely abolished the enzyme activity, indicating that these residues are essential for catalytic activity.
authors.affiliation: Eco-Friendly Bio-material Research Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 56212, Republic of Korea.
authors.forename: Jin-A
authors.initials: JA
authors.lastname: Ko
authors.affiliation: Department of Food Science & Technology and BK21 Plus Program, Bio-enery Research Center, Chonnam Natioanl University, Gwangju 61186, Republic of Korea.
authors.forename: Seung-Hee
authors.initials: SH
authors.lastname: Nam
authors.affiliation: Research Institute of Food Industrialization, Institutes of Green Bio Science & Technology, Seoul National University, Pyeongchang 25354, Republic of Korea.
authors.forename: Doman
authors.initials: D
authors.lastname: Kim
authors.affiliation: Department of Biotechnology, Chonnam National University, Gwangju 61186, Republic of Korea.
authors.forename: Jun-Ho
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Food Science & Technology and BK21 Plus Program, Bio-enery Research Center, Chonnam Natioanl University, Gwangju 61186, Republic of Korea.
authors.forename: Young-Min
authors.initials: YM
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D002855:Chromatography, Thin Layer; D003910:Dextranase / Q000737:chemistry* / Q000378:metabolism; D003911:Dextrans / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D056507:Paenibacillus / Q000737:chemistry / Q000201:enzymology*; D011487:Protein Conformation
chemical_list: D000596:Amino Acids; D003911:Dextrans; D003910:Dextranase
keywords: carboxyl group; catalytic amino acids; chemical modification; dextranase