Structural explanation for the acquisition of glycosynthase activity

Masafumi Hidaka, Shinya Fushinobu, Yuji Honda, Takayoshi Wakagi, Hirofumi Shoun, Motomitsu Kitaoka

研究成果: Article査読

18 被引用数 (Scopus)

抄録

Glycosynthases are engineered glycoside hydrolases (GHs) that catalyse the synthesis of glycoside from glycosyl-fluoride donors and suitable acceptors. We have determined five crystal structures of the glycosynthase mutants reducing-end xylose-releasing exo-oligoxylanase, an inverting GH, that exhibit various levels of glycosynthetic activities. At the active site of the Y198F mutant, the most efficient glycosynthase, a water molecule is observed at the same position as nucleophilic water (NW) in the parent enzyme, and the loss of the fixation of the direction of the lone pair of water molecules in the mutant drastically decreases hydrolytic activity. Water molecules were also observed at each active site of the general base mutant, but they were shifted 1.0-3.0 Å from the NW in the wild type. Their positions exhibited a strong correlation with the strength of glycosynthase activity. Here, we propose that a structural prerequisite for the sufficient glycosynthase reaction is the presence of a water molecule at the NW position, and mutation at the NW holder provides a general strategy for inverting GHs. The idea on the position of a water molecule may also be applicable to the design of efficient glycosynthases from retaining GHs.

本文言語English
ページ(範囲)237-244
ページ数8
ジャーナルJournal of biochemistry
147
2
DOI
出版ステータスPublished - 2010 2
外部発表はい

ASJC Scopus subject areas

  • 生化学
  • 分子生物学

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