Conversion of inverting glycoside hydrolases into catalysts for synthesizing glycosides employing a glycosynthase strategy.doc

1、TIGG TemplateConversion of inverting glycoside hydrolases into catalysts for synthesizing glycosides employing a glycosynthase strategy化反転型加水分解酵素合成触媒変換Kitaoka, Motomitsu1; Honda, Yuji1,2; Fushinobu, Shinya3; Hidaka, Masafumi1,3; Katayama, Takane2; and Yamamoto, Kenji41National Food Research Institut

2、e, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305-8642, Japan2Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Japan3Department of Biotechnology, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan4Division of Integrated Life Science, Graduate School of

3、Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, JapanFax: 81-29-838-7321, E-mail: mkitaokaaffrc.go.jp(Received Accepted)Key Words: glycosynthase, inverting hydrolase, glycosylfluoride, Hehre resynthesis-hydrlysis mechanism, reducing end xylose-releasing exo-oligoxylanase, -fucosidase.Abstrac

4、tReducing-end xylose-releasing exo-oligoxylanase (Rex, EC. 3.2.1.156) is an inverting xylanolytic enzyme, belonging to the glycoside hydrolase (GH) family 8, which hydrolyzes xylooligosaccharides to release xylose (X1) from its reducing end. Rex hydrolyzes -xylobiosyl fluoride (-X2F) to yield xylobi

5、ose (X2) only in the presence of X1, confirming the Hehre resynthesis-hydrolysis mechanism. A library of mutant Rex at the catalytic base (D263) was constructed by saturation mutagenesis, in which D263C accumulated the highest level of xylotriose (X3) from -X2F and X1. However, F releasing activitie

6、s of the mutants were much less than that of the wild type. Next, Y198 residue of Rex that forms a hydrogen bond with nucleophilic water was substituted with phenylalanine, causing a marked decrease in hydrolytic activity and a small increase in the F releasing activity from -X2F in the presence of

7、X1. Y198F of Rex accumulated more product during the glycosynthase reaction than D263C. Recently, an inverting -1,2-fucosidase belonging to GH95 was converted into glycosynthase by mutating a catalytic base residue. In both cases, the catalytic base should be intact. A. IntroductionEnzymatic synthes

8、is of glycosides (1-6) is a key topic in glycotechnology and does not require complicated organic synthesis procedures such as selective protection and deprotection of the hydroxyl group. Utilization of hydrolytic enzymes such as glycoside hydrolase, B. The Hehre resynthesis-hydrolysis reactionHehre

9、 and co-workers reported interesting phenomena associated with inverting GHs. Previously, they reported that -amylase, an inverting GH, hydrolyzed - and -maltosyl fluorides to maltose and F (13). It should be noted that C. GlycosynthaseThe mutant GHs that catalyze the synthesis of glycosides from th

10、e glycosyl fluoride of the opposite anomer are called glycosynthases (Fig. 3). D. A model of inverting GH Reducing end xylose-releasing exo-oligoxylanase (Rex) (69-71) GH8 is a family of inverting hydrolases whose members are mainly cellulase and chitosanase. In addition, endo -1,4 xylanase (pXyl),

11、belonging to this family, was found in a culture supernatant of Pseudoalteromonas haloplanktis and characterized (72). 還元末端遊離(Rex, EC. 3.2.1.156)、糖加水分解酵素属反転型分解酵素、糖還元末端(X1)遊離反応触媒。RexX1存在下、(-X2F)加水分解、Hehre-合成-加水分解反応示。一般塩基触媒残基(D263)変異酵素作製、活性-X2FX1(X3)最良蓄積D263C変異酵素選抜。、D263C素遊離活性親酵素低。次、一般塩基触媒残基共求核試薬水保持残基

12、Y198変異、素遊離活性低下加水分解活性大幅低下見。RexY198F変異酵素D263CX3蓄積多。最近、糖加水分解酵素属1,2化報告。場合、一般塩基触媒残基相互作用別残基変異入有効。両者、一般塩基触媒残基変異方良。有機合成、水酸基選択的保護脱保護最煩雑。酵素用合成(1-6)、往選択的保護脱保護回避、糖鎖工学重要要素一数。糖加水分解酵素(GH)、中略Hehre-再合成分解反応Hehre年代反転型加水分解酵素興味深現象報告。現象、-素加水分解最初報告(13)。注目、中略反対型基質合成変異加水分解酵素呼(図3)。中略還元末端遊離(Rex)：反転型加水分解酵素(69-71)GH8、主構成反転型加水分解

13、酵素。加、Pseudoalteromonas haloplanktis由来1,4-(pXyl)報告(72)。中略 References1.Nishimoto, M., and Kitaoka, M. (2007) Biosci. Biotechnol. Biochem. 71, 2101-21042.Totani, K., Yasutake, N., Murata, T., and Usui, T. (2004) Trends Glycosci. Glycotechnol. 16, 383-3923.Liu, Z., Lu, Y., Zhang, J., Pardee, K., and Wan

14、g, P. G. (2003) Appl. Environ. Microbiol. 69, 2110-21154.Koizumi, S. (2003) Trends Glycosci. Glycotechnol. 15, 65-745.Kitaoka, M., and Hayashi, K. (2002) Trends Glycosci. Glycotechnol. 14, 35-506.Murata, T., and Usui, T. (2000) Trends in Glycosci. Glycotechnol. 12, 161-1747.Henrissat, B., and Bairoc

15、h, A. (1996) Biochem. J. 316, 695-6968.Davies, G., and Henrissat, B. (1995) Structure 3, 853-859Examples of Figure, Scheme, and Table.Fig. 2. The Hehre resynthsis-hydrolysis reactionA. The reaction mechanism of inverting GH described as glucoamylase. B. The reaction mechanism of the Hehre resynthesi

16、s-hydrolysis reaction.Table I. List of enzymes reported to exhibit the Hehre resynthesis-hydrolysis reaction.EnzymeFamilyGlycoside hydrolyzedWrong fluoride usedDonor acts as acceptorreferences-amylase(GH14)-maltoside-maltosyl fluorideyes13glucoamylase(GH15)-glucoside-glucosyl fluorideyes22trehalaseNot assigned-glucoside-glucosyl fluorideno19,20-x