Catalytic Mechanism of Retaining α-Galactosidase Belonging to Glycoside Hydrolase Family 97

Okuyama, Masayuki; Kitamura, Momoyo; Hondoh, Hironori; Kang, Min-Sun; Mori, Haruhide; Kimura, Atsuo; Tanaka, Isao; Yao, Min

doi:10.1016/j.jmb.2009.07.068

Cited by 29 publications

(35 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…4C). 11 The reaction with maltose as the acceptor formed one major and two minor trisaccharide 12 (ESI-MS: m/z 527.16 for C18H32O16 + Na + ), and three minor tetrasaccharide (ESI-MS: m/z 689.21 13 for C24H42O21 + Na + ) products. The major trisaccharide was subjected to 1 H-NMR analysis.…”

mentioning

confidence: 99%

“…This compound was formed when the 9 hydroxy group at C6 of the glucose moiety of non-reducing end of cellobiose attacked the 10 anomeric carbon of the β-galactosyl formate intermediate. 11 The glycosidic linkages of the transfer products using PNP glucosides as acceptor 12 molecules were examined by methylation analysis. The analyses suggested that reactions formed 13 α-(1→6) glycosidic linkages.…”

mentioning

confidence: 99%

“…These acceptors 9 followed equation 2 with the exception of galactose, with no increase in the velocity of fluoride 10 release observed for galactose. The constants α, β, and γ for the glycosyl acceptors are 11 summarized in Table 3. 12 …”

mentioning

confidence: 99%

“…8B). In the 21 ligand-free wild-type enzyme [11], the calcium ion was bound to three glutamate residues 22 (Glu174, Glu464, and Glu470) and four water molecules with a pentagonal bipyramid geometry. 23 In the BtGH97b-D415G-Gal-Lac complex, three of the four water molecules were replaced by 24 hydroxyl groups and the α-galactosidic oxygen of Gal-Lac (Fig.…”

mentioning

confidence: 99%

“…Discussion 10 It is difficult to use an orthodox glycosynthase to synthesize an α-galactoside because of the 11 instability of β-galactosyl fluoride. In this study, we tested other known procedures for making α- 12 galactosides using a catalytic nucleophile mutant of GH97 α-galactosidase, BtGH97b-D415G.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Efficient synthesis of α‐galactosyl oligosaccharides using a mutant Bacteroides thetaiotaomicron retaining α‐galactosidase (BtGH97b)

et al. 2017

Self Cite

View full text Add to dashboard Cite

Abbreviations: α-GalF, α-galactopyranosyl fluoride; Gal-Lac, β-lactosyl α-D-galactopyranoside; 20 GH, glycoside hydrolase family; HMBC, heteronuclear multiple bond correlation; 21 high-performance anion exchange chromatography-pulsed amperometric detection. The preparation of a glycosynthase, a catalytic nucleophile mutant of a glycosidase, is a well-5 established strategy for the effective synthesis of glycosidic linkages. However, glycosynthases 6 derived from α-glycosidases can give poor yields of desired products because they require 7 generally unstable β-glycosyl fluoride donors. Here, we investigate a transglycosylation catalyzed 8 by a catalytic nucleophile mutant derived from a glycoside hydrolase family (GH) 97 α-9 galactosidase, using more stable β-galactosyl azide and α-galactosyl fluoride donors. The mutant 10 enzyme catalyzes the glycosynthase reaction using β-galactosyl azide and α-galactosyl transfer 11 from α-galactosyl fluoride with assistance of external anions. Formate was more effective at 12 restoring transfer activity than azide. Kinetic analysis suggests that poor transglycosylation in the 13 presence of the azide is because of low activity of the ternary complex between enzyme, β-14 galactosyl azide, and acceptor. A three-dimensional structure of the mutant enzyme in complex 15 with the transglycosylation product, β-lactosyl α-D-galactoside, was solved to elucidate the 16 ligand-binding aspects of the α-galactosidase. Subtle differences at the β→α loops 1, 2, and 3 of 17 the catalytic TIM barrel of the α-galactosidase from those of a homologous GH97 α-glucoside 18 hydrolase seem to be involved in substrate recognitions. In particular, the Trp residues in β→α 19 loop 1 have separate roles. Trp312 of the α-galactosidase appears to exclude the equatorial 20 hydroxy group at C4 of glucosides, whereas the corresponding Trp residue in the α-glucoside 21 hydrolase makes a hydrogen bond with this hydroxy group. The mechanism of α-galactoside-22 recognition is conserved among GH27, 31, 36, and 97 α-galactosidases. 23 24

show abstract

mentioning

confidence: 99%