The β-N-Acetylhexosaminidase in the Synthesis of Bioactive Glycans: Protein and Reaction Engineering

Bojarová, Pavla; Кулик, Н. В.; Hovorková, Michaela; Slámová, Kristýna; Křen, Vladimı́r

doi:10.3390/molecules24030599

Cited by 25 publications

(45 citation statements)

References 37 publications

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“…However, whereas other hydrolytic enzymes such as proteases, esterases, or lipases can cope with even non-aqueous systems (a w < 0.01), it appears that glycosidases require at least a w ≥ 0.9 to be active [129]. Nevertheless, lowering of a w has been applied for many GHs to increase their trans-glycosylation efficiency [129][130][131][132][133][134][135][136], including GH20 hexosaminidases [21,24,46,54,56,61,62,73,87,88,[90][91][92][95][96][97]100,101,[103][104][105][106]115,116,[118][119][120].…”

Section: Additives Increasing Trans-glycosylationmentioning

confidence: 99%

“…However, there are only a few studies, in which DMSO (5%-20% (v/v)) has been present during GH20-catalyzed trans-glycosylation (Tables 5, 9 and 10) [24,101,[103][104][105]. Especially for the fungal enzymes it seems to be more common to add acetonitrile (MeCN, 5%-45% (v/v)) as co-solvent to increase yields (Tables 2-4 and Table 10) [54,56,61,88,[90][91][92][95][96][97]. Other reported co-solvents are N,N-dimethylformamide (DMF, 5% (v/v)) [73,106], dioxane (20% (v/v)) [56], 1,3-butanediol and 1,2,4-butanetriol (8% (v/v)) [109].…”

Section: Co-solventsmentioning

confidence: 99%

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

et al. 2020

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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.

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Section: Additives Increasing Trans-glycosylationmentioning

confidence: 99%

Section: Co-solventsmentioning

confidence: 99%

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

et al. 2020

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“…p-coumaric or ferulic acid (300 mM), 30 mM p-nitrophenyl β-N-acetylglucosaminide (30 mM), McIlvaine buffer pH 5/40% (v/v) acetonitrile and the respective β-N-acetylhexosaminidase (1.5 U/mL). Fungal extracellular β-N-acetylhexosaminidases with a good transglycosylation potential [32] from the following sources were tested: Acremonium persicinum CCF 1850, Aspergillus oryzae CCF 1066, Fusarium oxysporum CCF 371, Penicillium chrysogenum CCF 1269, P. oxalicum CCF 2315, Talaromyces flavus CCF 2686, Trichoderma harzianum CCF 2687. Reactions were incubated at 35°C and 850 rpm for 24 hours and monitored by TLC (propan-2-ol:H 2 O:NH 4 OH aq.…”

Section: Glycosylation Of P-coumaric and Ferulic Acid With Hexosaminimentioning

confidence: 99%

Glycosidase‐Catalyzed Synthesis of Glycosyl Esters and Phenolic Glycosides of Aromatic Acids

et al. 2019

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Phenolic glycosides occur naturally in many plants and as such are often present in the human diet. Their isolation from natural sources is usually laborious due to their presence in complex matrices. Their chemical and enzymatic syntheses have been found complex, time-consuming, and costly, yielding only small amounts of glycosylated products. In quest of a convenient biocatalytic route to structurally complex phenolic glycosides, we discovered that the rutinosidase from Aspergillus niger not only efficiently converts hydroxylated aromatic acids (e. g. coumaric and ferulic acids) into the respective phenolic rutinosides, but surprisingly also catalyzes the formation of the respective glycosyl esters. We report here the results of a systematic study presenting the unique synthesis of naturally occurring glycosyl esters and phenolic glycosides accomplished by glycosidase catalysis. A panel of aromatic acids was tested as glycosyl acceptors and the crucial structural features required for the formation of glycosyl esters were identified. In the light of the present structure-activity relationship study, a plausible reaction mechanism was proposed. All the products were fully structurally characterized by NMR and MS. Scheme 2. Glycosylation of (E)-p-coumaric acid (3). Scheme 3. Proposed mechanism of rutinosylation of hydroxycinnamic acids at the carboxy moiety.

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“…Since GlcA (4) and MurNAc (3) were not recognized as acceptors by the wild-type enzyme, they were subjected to testing using the mutant enzyme Tf Hex Y470H with suppressed hydrolytic activity and increased transglycosylation potential [13]. Besides free monosaccharides, a series of GlcA and MurNAc glycosides [16] functionalized at the anomeric position (11-15, Figure 3) were also employed as acceptors in the transglycosylation reaction mediated by this biocatalyst to test the influence of the anomeric effect on the glycosylation process.…”

Section: Glycosylation Reaction Catalyzed By Tfhex Y470h: Screening Omentioning

confidence: 99%

“…In this work, we have focused on the β-N-acetylhexosaminidase from Talaromyces flavus CCF 2686 (Tf Hex), a representative of transglycosylating β-N-acetylhexosaminidases, due to its great substrate flexibility, which enhances its potential in synthetic reactions, and the availability of the molecular model of this enzyme. In addition, site-directed mutagenesis has allowed the improvement of its synthetic properties, generating a considerable increase in its transglycosylation capability through the substitution of the enzyme active site residue tyrosine 470 by histidine [11,13]. The Tf Hex Y470H mutant has been demonstrated as an efficient synthetic tool [14].…”

Section: Introductionmentioning

confidence: 99%

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

García‐Oliva

Hoyos

Petrásková

et al. 2019

IJMS

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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.

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The β-N-Acetylhexosaminidase in the Synthesis of Bioactive Glycans: Protein and Reaction Engineering

Cited by 25 publications

References 37 publications

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

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

Glycosidase‐Catalyzed Synthesis of Glycosyl Esters and Phenolic Glycosides of Aromatic Acids

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

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