HUSCAP logo Hokkaido Univ. logo

Hokkaido University Collection of Scholarly and Academic Papers >
Graduate School of Agriculture / Faculty of Agriculture >
Peer-reviewed Journal Articles, etc >

Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach

Files in This Item:
JBC287-24_19927-19935.pdf517.67 kBPDFView/Open
Please use this identifier to cite or link to this item:http://hdl.handle.net/2115/51765

Title: Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach
Authors: Kim, Young-Min Browse this author
Kiso, Yoshiaki Browse this author
Muraki, Tomoe Browse this author
Kang, Min-Sun Browse this author
Nakai, Hiroyuki Browse this author
Saburi, Wataru Browse this author →KAKEN DB
Lang, Weeranuch Browse this author
Kang, Hee-Kwon Browse this author
Okuyama, Masayuki Browse this author →KAKEN DB
Mori, Haruhide Browse this author →KAKEN DB
Suzuki, Ryuichiro Browse this author
Funane, Kazumi Browse this author
Suzuki, Nobuhiro Browse this author
Momma, Mitsuru Browse this author
Fujimoto, Zui Browse this author
Oguma, Tetsuya Browse this author
Kobayashi, Mikihiko Browse this author
Kim, Doman Browse this author
Kimura, Atsuo Browse this author →KAKEN DB
Keywords: dextranase
cycloisomaltooligosaccharide
glycoside hydrolase family 66
catalytic residues
Issue Date: 8-Jun-2012
Publisher: American Society for Biochemistry and Molecular Biology
Journal Title: Journal of Biological Chemistry
Volume: 287
Issue: 24
Start Page: 19927
End Page: 19935
Publisher DOI: 10.1074/jbc.M111.339036
Abstract: A novel endo-dextranase (PsDex) from Paenibacillus sp. was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization=of 7-14 from dextran. The 1,696 amino-acid sequence belonging to the glycosyl hydrolase family (GH) 66 has a long insertion (632 residues; Thr451-Val1082), a portion of which shares identity (35% at Ala39-Ser1304 of PsDex) with Pro32-Ala755 of CI glucanotransferase (CITase), a GH 66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val837-Met932 for PsDex and Tyr404-Tyr492 for CITase), similar to carbohydrate-binding module 35, was not found in other endo-dextranases (Dexs) devoid of CITase activity. These results support the classification of GH 66 enzymes into 3 types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala39-Ser1304) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH 66 enzymes possess 4 conserved acidic residues (Asp189, Asp340, Glu412, and Asp1254 of PsDex) of catalytic candidates. Their amide-mutants decreased activity (1/1500- 1/40,000-time), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using α-isomaltotetraosyl fluoride with NaN3. D340G or E412Q formed a β- or α-isomaltotetraosyl azide, respectively, strongly indicating that Asp340 and Glu412 are nucleophile and acid/base-catalyst, respectively. Interestingly, D189A synthesized small-sized dextran from α-isomaltotetraosyl fluoride in the presence of NaN3.
Rights: This research was originally published in Journal of Biological Chemistry. Young-Min Kim; Yoshiaki Kiso; Tomoe Muraki; Min-Sun Kang; Hiroyuki Nakai; Wataru Saburi; Weeranuch Lang; Hee-Kwon Kang; Masayuki Okuyama; Haruhide Mori; Ryuichiro Suzuki; Kazumi Funane; Nobuhiro Suzuki; Mitsuru Momma; Zui Fujimoto; Tetsuya Oguma; Mikihiko Kobayashi; Doman Kim; Atsuo Kimura. Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach. Journal of Biological Chemistry. 2012; 287:19927-19935. © the American Society for Biochemistry and Molecular Biology.
Type: article (author version)
URI: http://hdl.handle.net/2115/51765
Appears in Collections:農学院・農学研究院 (Graduate School of Agriculture / Faculty of Agriculture) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 木村 淳夫

Export metadata:

OAI-PMH ( junii2 , jpcoar )


 

Feedback - Hokkaido University