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)
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