open access

https://hdl.handle.net/2115/51765

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

英語

catalytic residues

2012-06-08

American Society for Biochemistry and Molecular Biology

Journal of Biological Chemistry

287(24)

19927-19935

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.

学術雑誌論文

Accepted Manuscript

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.

0021-9258