2001年
超好熱性古細菌 Thermococcus litoralis 由来4-α-グルカノトランスフェラーゼの反応機構と構造解析
応用糖質科学
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- 巻
- 48
- 号
- 2
- 開始ページ
- 171
- 終了ページ
- 175
- 記述言語
- 日本語
- 掲載種別
- DOI
- 10.5458/jag.48.171
- 出版者・発行元
- The Japanese Society of Applied Glycoscience
Thermococcus litoralis 4-α-glucanotransferase (GTase) belongs to family 57 of glycosyl hydro-lases and catalyzes the disproportionation and cycloamylose synthesis reactions. Using 3-ketobutylidene-β-2-chloro-4-nitrophenyl-maltopentaoside (3 KBG 5 CNP) as a donor and glucose as an acceptor, we showed that the disproportionation reaction of 4-α-glucanotransferase involves a Ping-Pong Bi Bi mechanism. Based on this reaction mechanism, we trapped the glycosyl-enzyme intermediate by treating the enzyme with 3 KBG 5 CNP in the absence of an acceptor. MALDITOFMS and PSD analysis of a peptic digest of the intermediate and great decrease in activity of the E 123 Q mutant enzyme indicated that completely conserved Glu 123 was the catalytic amino acid. Next, we have determined the structure of GTase, the first structure of the family 57 of glycosyl hydrolases, at 2.8 Å resolution. The structure of GTase is composed of three domains. N-terminal domain (domain A) contains two catalytic residues and has a novel pseudo-TIM barrel fold. C-terminal domain (domain C) is mainly made of β-strands arranged in two layered β-sheet sandwich. Domain B is located between domains A and C, and consists of α- and 310-helices. Active site cleft lies between domains A and B. The cleft is partially covered with aromatic residues to form a tunnel-like shape, which probably plays an important role in the formation of large cyclic glucans.
- リンク情報
- ID情報
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- DOI : 10.5458/jag.48.171
- ISSN : 1340-3494
- CiNii Articles ID : 10008252642
- CiNii Books ID : AN10453916