β-N-Acetylhexosaminidases—the wizards of glycosylation

Bojarová, Pavla; Bruthans, Jan; Křen, Vladimı́r

doi:10.1007/s00253-019-10065-0

Cited by 23 publications

(17 citation statements)

References 102 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In contrast, the reaction performed with the pNP-GalNAc donor and GalNAc (5) acceptor ( Table 1, entry II), exclusively resulted in the β(1-6) regioisomer (8, Figure 2) in a 31% yield. As a rule of thumb, the axial galacto-position is considered practically impossible to glycosylate with β-N-acetylhexosaminidases [7]. This study demonstrates the high regioselectivity of β-N-acetylhexosaminidase-catalyzed glycosylation, depending on the configuration of the C-4 acceptor hydroxyl ( Figure 2).…”

Section: Glycosylation Reaction Catalyzed By Tf Hex Wt: Screening Of mentioning

confidence: 71%

“…As exemplified above, Tf Hex WT is an excellent tool in carbohydrate synthesis [7]. For the aim of exploring the spectrum of acceptor substrates of this enzyme, we first decided to test its activity in the transglycosylation reaction involving pNP-GlcNAc or pNP-GalNAc as glycosyl donors and various natural monosaccharides of the glucoand galacto-configuration as acceptors, namely GlcNAc (1), Glc (2), MurNAc (3), GlcA (4), Gal (6), and GalNAc (5) (Figure 1).…”

Section: Glycosylation Reaction Catalyzed By Tf Hex Wt: Screening Of mentioning

confidence: 99%

“…First, reactions with acceptor substrates 1-6 were conducted at an analytical scale (200 µL) and in case product formation was detected, reactions were scaled up to a 3 mL volume. The outcome of respective transglycosylation reactions is shown in Figures S1-S6 As exemplified above, TfHex WT is an excellent tool in carbohydrate synthesis [7]. For the aim of exploring the spectrum of acceptor substrates of this enzyme, we first decided to test its activity in the transglycosylation reaction involving pNP-GlcNAc or pNP-GalNAc as glycosyl donors and various natural monosaccharides of the gluco-and galacto-configuration as acceptors, namely GlcNAc (1), Glc (2), MurNAc (3), GlcA (4), Gal (6), and GalNAc (5) ( Figure 1).…”

Section: Glycosylation Reaction Catalyzed By Tf Hex Wt: Screening Of mentioning

confidence: 99%

“…The broad range of substrates accepted makes these enzymes ideal synthetic tools with non-natural substrates [7]. In this context, β-N-acetylhexosaminidases from fungi have been demonstrated as attractive catalysts for this purpose [8][9][10].…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation

García‐Oliva

Hoyos

Petrásková

et al. 2019

IJMS

Self Cite

View full text Add to dashboard Cite

Fungal β-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The β-N-acetylhexosaminidase from Talaromyces flavus is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to substrate modifications. It can be heterologously produced in Pichia pastoris in a high yield. The mutation of the Tyr470 residue to histidine greatly enhances its transglycosylation capability. The aim of this work was to identify the structural requirements of this model β-N-acetylhexosaminidase for its transglycosylation acceptors and formulate a structure–activity relationship study. Enzymatic reactions were performed using an activated glycosyl donor, 4-nitrophenyl N-acetyl-β-d-glucosaminide or 4-nitrophenyl N-acetyl-β-d-galactosaminide, and a panel of glycosyl acceptors of varying structural features (N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose, N-acetylmuramic acid, and glucuronic acid). The transglycosylation products were isolated and structurally characterized. The C-2 N-acetamido group in the acceptor molecule was found to be essential for recognition by the enzyme. The presence of the C-2 hydroxyl moiety strongly hindered the normal course of transglycosylation, yielding unique non-reducing disaccharides in a low yield. Moreover, whereas the gluco-configuration at C-4 steered the glycosylation into the β(1-4) position, the galacto-acceptor afforded a β(1-6) glycosidic linkage. The Y470H mutant enzyme was tested with acceptors based on β-glycosides of uronic acid and N-acetylmuramic acid. With the latter acceptor, we were able to isolate and characterize one glycosylation product in a low yield. To our knowledge, this is the first example of enzymatic glycosylation of an N-acetylmuramic acid derivative. In order to explain these findings and predict enzyme behavior, a modeling study was accomplished that correlated with the acquired experimental data.

show abstract

Section: Glycosylation Reaction Catalyzed By Tf Hex Wt: Screening Of mentioning

confidence: 71%

Section: Glycosylation Reaction Catalyzed By Tf Hex Wt: Screening Of mentioning

confidence: 99%

Section: Glycosylation Reaction Catalyzed By Tf Hex Wt: Screening Of mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation

García‐Oliva

Hoyos

Petrásková

et al. 2019

IJMS

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, it is a general feature of many retaining GHs that other nucleophiles such as carbohydrates or alcohols are also accepted, which then leads to a glycosylated product (trans-glycosylation, Figure 2: R 1 H, R 2 H) [35]. Most of the CAZymes acting on GlcNAc residues follow this mechanism (e.g., chitinases (GH18), O-GlcNAcases (GH84), endo-β-N-acetylglucosaminidases (GH85)) [3,[36][37][38]. Only hexosaminidases from family GH3 [39] and the recently discovered GH136 lacto-N-biosidase LnbX from Bifidobacterium longum subsp.…”

Section: Gh20 β-N-acetylhexosaminidasesmentioning

confidence: 99%

β-N-Acetylhexosaminidases for Carbohydrate Synthesis via Trans-Glycosylation

et al. 2020

View full text Add to dashboard Cite

β-N-acetylhexosaminidases (EC 3.2.1.52) are retaining hydrolases of glycoside hydrolase family 20 (GH20). These enzymes catalyze hydrolysis of terminal, non-reducing N-acetylhexosamine residues, notably N-acetylglucosamine or N-acetylgalactosamine, in N-acetyl-β-D-hexosaminides. In nature, bacterial β-N-acetylhexosaminidases are mainly involved in cell wall peptidoglycan synthesis, analogously, fungal β-N-acetylhexosaminidases act on cell wall chitin. The enzymes work via a distinct substrate-assisted mechanism that utilizes the 2-acetamido group as nucleophile. Curiously, the β-N-acetylhexosaminidases possess an inherent trans-glycosylation ability which is potentially useful for biocatalytic synthesis of functional carbohydrates, including biomimetic synthesis of human milk oligosaccharides and other glycan-functionalized compounds. In this review, we summarize the reaction engineering approaches (donor substrate activation, additives, and reaction conditions) that have proven useful for enhancing trans-glycosylation activity of GH20 β-N-acetylhexosaminidases. We provide comprehensive overviews of reported synthesis reactions with GH20 enzymes, including tables that list the specific enzyme used, donor and acceptor substrates, reaction conditions, and details of the products and yields obtained. We also describe the active site traits and mutations that appear to favor trans-glycosylation activity of GH20 β-N-acetylhexosaminidases. Finally, we discuss novel protein engineering strategies and suggest potential “hotspots” for mutations to promote trans-glycosylation activity in GH20 for efficient synthesis of specific functional carbohydrates and other glyco-engineered products.

show abstract

How Site‐Directed Mutagenesis Boosted Selectivity of a Promiscuous Enzyme

et al. 2020

Self Cite

View full text Add to dashboard Cite

β-N-Acetylhexosaminidases (GH20; EC 3.2.1.52) are exo-glycosidases with a dual activity for cleaving both N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from glycostructures. This substrate promiscuity is a hurdle in the selective synthesis of N-acetylhexosamine oligosaccharides combining both GlcNAc and GalNAc units since there are hardly any GalNAc transferring enzymes available for synthetic applications. We present here site-directed mutagenesis of a synthetically potent promiscuous β-N-acetylhexosaminidase from Talaromyces flavus (TfHex), which, as a wild type, exhibits a GalNAcase/ GlcNAcase ratio of 1.2. On the basis of molecular modeling, we identified crucial amino acid residues responsible for its GalNAcase/GlcNAcase selectivity. Six site-directed mutants were prepared, heterologously expressed in Pichia pastoris, purified, and kinetically characterized. As a result, novel engineered enzymes with an up to 7-times higher selectivity for either GalNAc or GlcNAc substrates were obtained, preserving the favorable properties of the wild type TfHex, mainly its transglycosylation potential and tolerance to functional groups in the substrate molecule. The substrate selectivity and transglycosylation yield were further corroborated by reaction engineering. The new selective and synthetically capable enzymes were applied in the preparation of tailored N-acetylhexosamines.

show abstract

β-N-Acetylhexosaminidases—the wizards of glycosylation

Cited by 23 publications

References 102 publications

Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation

Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation

β-N-Acetylhexosaminidases for Carbohydrate Synthesis via Trans-Glycosylation

How Site‐Directed Mutagenesis Boosted Selectivity of a Promiscuous Enzyme

Contact Info

Product

Resources

About