5e1q
From Proteopedia
Mutant (D415G) GH97 alpha-galactosidase in complex with Gal-Lac
Structural highlights
Function[AGAL_BACTN] Galactosidase that is able to hydrolyze the alpha-1,6 disaccharide melibiose and the synthetic p-nitrophenyl alpha-galactoside substrate (pNP-Gal), with retention of the anomeric configuration. Does not hydrolyze DNP-Glc or pNP-Glc.[1] [2] Publication Abstract from PubMedThe preparation of a glycosynthase, a catalytic nucleophile mutant of a glycosidase, is a well-established strategy for the effective synthesis of glycosidic linkages. However, glycosynthases derived from alpha-glycosidases can give poor yields of desired products because they require generally unstable beta-glycosyl fluoride donors. Here, we investigate a transglycosylation catalyzed by a catalytic nucleophile mutant derived from a glycoside hydrolase family (GH) 97 alpha-galactosidase, using more stable beta-galactosyl azide and alpha-galactosyl fluoride donors. The mutant enzyme catalyzes the glycosynthase reaction using beta-galactosyl azide and alpha-galactosyl transfer from alpha-galactosyl fluoride with assistance of external anions. Formate was more effective at restoring transfer activity than azide. Kinetic analysis suggests that poor transglycosylation in the presence of the azide is because of low activity of the ternary complex between enzyme, beta-galactosyl azide, and acceptor. A three-dimensional structure of the mutant enzyme in complex with the transglycosylation product, beta-lactosyl alpha-d-galactoside, was solved to elucidate the ligand-binding aspects of the alpha-galactosidase. Subtle differences at the beta-->alpha loops 1, 2, and 3 of the catalytic TIM barrel of the alpha-galactosidase from those of a homologous GH97 alpha-glucoside hydrolase seem to be involved in substrate recognitions. In particular, the Trp residues in beta-->alpha loop 1 have separate roles. Trp312 of the alpha-galactosidase appears to exclude the equatorial hydroxy group at C4 of glucosides, whereas the corresponding Trp residue in the alpha-glucoside hydrolase makes a hydrogen bond with this hydroxy group. The mechanism of alpha-galactoside-recognition is conserved among GH27, 31, 36, and 97 alpha-galactosidases. This article is protected by copyright. All rights reserved. Efficient synthesis of alpha-galactosyl oligosaccharides using a mutant Bacteroides thetaiotaomicron retaining alpha-galactosidase (BtGH97b).,Okuyama M, Matsunaga K, Watanabe KI, Yamashita K, Tagami T, Kikuchi A, Ma M, Klahan P, Mori H, Yao M, Kimura A FEBS J. 2017 Jan 19. doi: 10.1111/febs.14018. PMID:28103425[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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