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

Abstract

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

Mutagenesis of Catalytic Nucleophile of β‐Galactosidase Retains Residual Hydrolytic Activity and Affords a Transgalactosidase

“…Surprisingly, all the catalytic nucleophile mutants exhibited a non-negligible residual hydrolytic activity with pNP-β-Gal substrate. This is in contrast to previous findings on other glycosynthases in the literature [12,17] -it has been generally accepted so far that this type of mutation leads to the complete abolishment of hydrolytic activity in retaining glycosidases. We also considered the issue of translational misincorporation [34] that might have resulted in the contamination of mutant enzyme samples by traces of WT enzyme.…”

“…When the nucleophile (Asp or Glu) is replaced by a non-nucleophilic residue, it affords a glycosynthase -a hydrolytically inactive enzyme unable to form the glycosyl-enzyme intermediate. [12,16,17] It is possible to rescue the activity of glycosynthases using an activated substrate of the 'correct' anomeric configuration by adding smallmolecule exogenous nucleophiles (sodium azide or sodium…”

Loop engineering of an α-1,3/4-l-fucosidase for improved synthesis of human milk oligosaccharides

“…The only previously reported engineering of BbAfcB is its transformation into an α-L-fucosynthase, giving an LNFP II yield of 41% when using 34 times more enzyme than in the current work [22]. While this strategy is appealing due to its simple, generic nature, especially αglycosynthases struggle with low donor substrate stability [22,44]. GH29B α-1,3/4-L-fucosidases are particularly appealing in terms of engineering for improved transfucosylation activityand for use in transfucosylation in generalsince they appear to maintain their high regioselectivity in the transfucosylation reaction, while the GH29A α-Lfucosidases often do not [10,12,22].…”

Synthesis of Glycosides by Glycosynthases

“…Cobucci-Ponzano et al reported the effective conversion of β- d -galactopyranosyl azide ( 35 , βGalN 3 ) to α-galacto-oligosaccharides by the glycosynthase TmGalA D327G derived from T. maritima [ 40 ]. The enzyme produced galacto-oligosaccharides in good yields and the method was recently expanded by Okuyama et al to the in situ formation of a β- d -galactosyl formate donor ( 36 ), which exhibited a higher transglycosylation rate compared to the azide donor ( 35 , Scheme 6 ) [ 41 ]. The method allowed the galactosylation of carbohydrates such as glucose (α-1,1-β), xylose (α-1,4), maltose (α-1,1-β), cellobiose (α-1,1-β; α-1,6), lactose (α-1,1-β), and pNP derivatives of glucose (pNPGlc, 37 ), and mannose (pNPMan, 6 ; exclusively α-1,6) in yields ranging from 75 to 95%.…”

“… In situ formation of the glycosyl donors βGalN 3 ( 35 ) or βGal formate ( 36 ) by incubation of αGalF ( 2 ) with glycosynthase BtGH97b D415G the additional external nucleophiles sodium azide of formate [ 41 ]. The in situ produced donor can then be subsequently transferred to a suitable acceptor (R 2 OH).…”