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Glycobiology Advance Access originally published online on February 9, 2008
Glycobiology 2008 18(4):325-330; doi:10.1093/glycob/cwn011
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© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Alternative strategy for converting an inverting glycoside hydrolase into a glycosynthase

Yuji Honda2, Shinya Fushinobu3, Masafumi Hidaka4, Takayoshi Wakagi3, Hirofumi Shoun3, Hajime Taniguchi2 and Motomitsu Kitaoka1,4

2 Ishikawa Prefectural University, 1-308, Suematsu, Nonoichi, Ishikawa 921-8836
3 Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657
4 National Food Research Institute, 2-1-12, Kannondai, Tsukuba, Ibaraki 305-8642, Japan

1 To whom correspondence should be addressed: Tel: +81-29-838-8071; Fax: +81-29-838-7321; e-mail: mkitaoka{at}affrc.go.jp

Received on October 3, 2007; revised on December 26, 2007; accepted on February 3, 2008

The tyrosine residue Y198 is known to support a nucleophilic water molecule with the general base residue, D263, in the reducing-end xylose-releasing exo-oligoxylanase (Rex). A mutation in the tyrosine residue changing it into phenylalanine caused a drastic decrease in the hydrolytic activity and a small increase in the F– releasing activity from {alpha}-xylobiosyl fluoride in the presence of xylose. In contrast, mutations at D263 resulted in the decreased F– releasing activity. As a result of the high F– releasing activity and low hydrolytic activity, Y198F of Rex accumulates a large amount of product during the glycosynthase reaction. We propose a novel method for producing a glycosynthase from an inverting glycoside hydrolase by mutating a residue that holds the nucleophilic water molecule with the general base residue while keeping the general base residue intact.

Key words: general base / glycosyl fluoride / glycosynthase / inverting glycoside hydrolase / reducing-end xylose-releasing exo-oligoxylanase


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