It All Starts with a Sandwich: Identification of Sialidases with Trans-Glycosylation Activity

Nordvang, Rune Thorbjørn; Nyffenegger, Christian; Holck, Jesper; Jers, Carsten; Zeuner, Birgitte; Sundekilde, Ulrik Kræmer; Meyer, Anne S.; Mikkelsen, Jørn Dalgaard

doi:10.1371/journal.pone.0158434

Cited by 19 publications

(16 citation statements)

References 32 publications

(52 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As hypothesized above, XyG accessibility may be limited for TmαFuc by the loop structure at the entrance to its active site ( Figure S3g). As previously shown for sialidases, transglycosylation activity is not only linked to how open the active site binding pocket is, but also to specific interactions inside or near the active site [48]. Indeed, in the current work both donor and acceptor specificities of each α-L-fucosidase seem to influence their transfucosylation potential significantly.…”

Section: Fgfco1 Catalysed Formation Of 2'fl Reaching a Molar Yield Basupporting

confidence: 64%

Substrate specificity and transfucosylation activity of GH29 α-l-fucosidases for enzymatic production of human milk oligosaccharides

et al. 2018

Self Cite

View full text Add to dashboard Cite

Human milk oligosaccharides (HMOs) constitute a unique family of bioactive lactose-based molecules present in human breast milk. HMOs are of major importance for infant health and development but also virtually absent from bovine milk used for infant formula. Among the HMOs, the fucosylated species are the most abundant. Transfucosylation catalysed by retaining α-l-fucosidases is a new route for manufacturing biomimetic HMOs. Seven α-l-fucosidases from glycosyl hydrolase family 29 were expressed, characterized in terms of substrate specificity and thermal stability, and shown to be able to catalyse transfucosylation. The α-l-1,3/4-fucosidase CpAfc2 from Clostridium perfringens efficiently catalysed the formation of the more complex human milk oligosaccharide structure lacto-N-fucopentaose II (LNFP II) using 3-fucosyllactose as fucosyl donor and lacto-N-tetraose as acceptor with a 39% yield. α-l-Fucosidases FgFCO1 from Fusarium graminearum and Mfuc5 from a soil metagenome were able to catalyse transfucosylation of lactose using citrus xyloglucan as fucosyl donor. FgFCO1 catalysed formation of 2'-fucosyllactose, whereas Mfuc5 catalysis mainly produced an unidentified, non-HMO fucosyllactose, reaching molar yields based on the donor substrate of 14% and 18%, respectively.

show abstract

Section: Fgfco1 Catalysed Formation Of 2'fl Reaching a Molar Yield Basupporting

confidence: 64%

Substrate specificity and transfucosylation activity of GH29 α-l-fucosidases for enzymatic production of human milk oligosaccharides

et al. 2018

Self Cite

View full text Add to dashboard Cite

show abstract

“…We observed that the total transglycosylation to hydrolysis (TG/HT) product ratio for the CelE-E316G-CBM3a construct was 40 to 140-fold higher than the values obtained for the control wild-type enzymes. Clearly our chimeric mutant is a highly efficient transglycosidase based on TG/HT metrics established for other CAZyme systems (Lundemo et al, 2013;Nordvang et al, 2016). Previous studies on another glucosidase have reported similar order of magnitude (70-fold) increase in TG/HT ratio but only after extensive mutagenesis and directed evolution of the wild-type enzymes (Kone et al, 2008), which further highlights the advantages offered by our current approach.…”

Section: Discussionsupporting

confidence: 52%

Carbohydrate binding domains facilitate efficient oligosaccharides synthesis by enhancing mutant catalytic domain transglycosylation activity

Bandi

Gonçalves

Pingali

et al. 2020

Preprint

View full text Add to dashboard Cite

Chemoenzymatic approaches using carbohydrate-active enzymes (CAZymes) offer a promising avenue for synthesis of glycans like oligosaccharides. Here, we report a novel chemoenzymatic route for cellodextrins synthesis employed by chimeric CAZymes, akin to native glycosyltransferases, involving the unprecedented participation of a 'noncatalytic' lectin-like or carbohydrate-binding domains (CBMs) in the catalytic step for glycosidic bond synthesis using cellobiosyl donor sugars as activated substrates. CBMs are often thought to play a passive substrate targeting role in enzymatic glycosylation reactions mostly via overcoming substrate diffusion limitations for tethered catalytic domains (CDs) but are not known to participate directly in any nucleophilic substitution mechanisms that impact the actual glycosyl transfer step. Our study provides evidence for the direct participation of CBMs in the catalytic reaction step for -glucan glycosidic bonds synthesis enhancing activity for CBM-based CAZyme chimeras by >140-fold over CDs alone. Dynamic intra-domain interactions that facilitate this poorly understood reaction mechanism were further revealed by small-angle X-ray scattering structural analysis along with detailed mutagenesis studies to shed light on our current limited understanding of similar transglycosylation-type reaction mechanisms. In summary, our study provides a novel strategy for engineering similar CBMbased CAZyme chimeras for synthesis of bespoke oligosaccharides using simple activated sugar monomers. Lombard et al., 2014), and still growing with newly sequenced genomes and metagenomes becoming readily available.CAZymes like GHs or GTs are further classified either as retaining or inverting type, depending on whether the stereochemistry at the anomeric carbon for the product (versus the reactant) is either retained or inverted (Davies and Henrissat, 1995), respectively. As first proposed by Koshland for glycosidases (Koshland, 1953), retaining enzymes require a classical two-step double displacement hydrolysis mechanism (i.e., SN2) with a glycosyl-enzyme intermediate (GEI) formed via a covalent bond between the cleaved substrate and the protein in the alternate orientation (e.g., GEI for retaining enzyme will have an -bond, as opposed to the -orientation of the reactant and product). While most native glycosidases catalyze the hydrolysis of glycosidic linkages, many GHs can also produce oligosaccharides following a competing transglycosylation mechanism if a suitably localized glycosyl acceptor group is present instead of a water molecule within the enzyme active site (Bissaro et al., 2015). Retaining GHs that show significant transglycosylation reactivity are also thought to utilize a similar SN2 type general mechanism, except that rather than nucleophilic attack by water, the attack is instigated by a hydroxyl oxygen on an acceptor sugar placed adjacent to the GEI complex (Figure 1).

show abstract

“…The binding pockets of HEX1 GTDDA and HEX1 GTEPG were much more shielded by the Trp residues than that of HEX1, so that the intermediate might be more protected against water molecules and, therefore, also against hydrolysis. The importance of such aromatic residues leading to a narrow substrate‐binding site for transglycosidase activity was also described for a GH33 sialidase …”

Section: Resultsmentioning

confidence: 96%

Loop Protein Engineering for Improved Transglycosylation Activity of a β‐N‐Acetylhexosaminidase

et al. 2018

Self Cite

View full text Add to dashboard Cite

Certain enzymes of the glycoside hydrolase family 20 (GH20) exert transglycosylation activity and catalyze the transfer of β-N-acetylglucosamine (GlcNAc) from a chitobiose donor to lactose to produce lacto-N-triose II (LNT2), a key human milk oligosaccharide backbone moiety. The present work is aimed at increasing the transglycosylation activity of two selected hexosaminidases, HEX1 and HEX2, to synthesize LNT2 from lactose and chitobiose. Peptide pattern recognition analysis was used to categorize all GH20 proteins in subgroups. On this basis, we identified a series of proteins related to HEX1 and HEX2. By sequence alignment, four additional loop sequences were identified that were not present in HEX1 and HEX2. Insertion of these loop sequences into the wild-type sequences induced increased transglycosylation activity for three out of eight mutants. The best mutant, HEX1 , had a transglycosylation yield of LNT2 on the donor that was nine times higher than that of the wild-type enzyme. Homology modeling of the enzymes revealed that the loop insertion produced a more shielded substrate-binding pocket. This shielding is suggested to explain the reduced hydrolytic activity, which in turn resulted in the increased transglycosylation activity of HEX1 .

show abstract

It All Starts with a Sandwich: Identification of Sialidases with Trans-Glycosylation Activity

Cited by 19 publications

References 32 publications

Substrate specificity and transfucosylation activity of GH29 α-l-fucosidases for enzymatic production of human milk oligosaccharides

Substrate specificity and transfucosylation activity of GH29 α-l-fucosidases for enzymatic production of human milk oligosaccharides

Carbohydrate binding domains facilitate efficient oligosaccharides synthesis by enhancing mutant catalytic domain transglycosylation activity

Loop Protein Engineering for Improved Transglycosylation Activity of a β‐N‐Acetylhexosaminidase

Contact Info

Product

Resources

About