Abstract
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.
Highlights
N-acetylhexosamines are important constituents in several biological and biochemically significant structures
When comparing the homology models of BbhI and the Hex1-GTEPG loop mutant by the AoHex enzyme and the many different mutants of this enzyme derived from Aspergillus oryzae, (Figure 7), we noticed that the mutated W805 in BbhI and the newly positioned R360 in Hex1-GTEPG are and more recently for other glycoside hydrolase family 20 (GH20) enzymes, in particular from Bifidobacterium bifidum
GH20 trans-glycosylation reactions, but reaction engineering involving various types of donor. Such an Arg residue might be responsible for modulating the water network or binding water in general, activation, high A:D ratio, high substrate concentration in general, lowering of aw by addition of cowhich leadssalts to lowered availability of water can for hydrolysis, as proposed foraltered solvents, or addition of cyclodextrins lead to increased yields or distinct catalytic mechanism of the GH20 β-N-acetylhexosaminidases, involving substrate-assisted catalysis in which the 2-acetamido group acts as an intramolecular nucleophile leading to formation of an oxazolinium ion intermediate, has proven uniquely useful as a blueprint for using oxazolineconjugated substrates for trans-glycosylation
Summary
N-acetylhexosamines are important constituents in several biological and biochemically significant structures. Glycans, oligo- and polysaccharides as well as other glycosylated molecules are synthesized by glycosyltransferases (GTs), which strictly require a nucleotide-activated derivative as synthesized by glycosyltransferases (GTs), which strictly require a nucleotide-activated derivative as substrate These enzymes are not very attractive for biocatalytic reactions, because the prices substrate. GH glycosylation reactions can be rather low, which leads to a mixture of the desired product and trans-glycosylases are in factSecondly, hydrolytic enzymes or are derived such. They are enzymes usually still undesired side products. Employed to increase trans-glycosylation activity and/or diminish hydrolytic activity in general
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