N-Acetylhexosamine oligosaccharides terminated with GalNAc act as selective ligands of galectin-3, a biomedically important human lectin. Their synthesis can be accomplished by β-N-acetylhexosaminidases (EC 3.2.1.52). Advantageously, these enzymes tolerate the presence of functional groups in the substrate molecule, such as the thiourea linker useful for covalent conjugation of glycans to a multivalent carrier, affording glyconjugates. β-N-Acetylhexosaminidases exhibit activity towards both N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) moieties. A point mutation of active-site amino acid Tyr into other amino acid residues, especially Phe, His, and Asn, has previously been shown to strongly suppress the hydrolytic activity of β-N-acetylhexosaminidases, creating enzymatic synthetic engines. In the present work, we demonstrate that Tyr470 is an important mutation hotspot for altering the ratio of GlcNAcase/GalNAcase activity, resulting in mutant enzymes with varying affinity to GlcNAc/GalNAc substrates. The enzyme selectivity may additionally be manipulated by altering the reaction medium upon changing pH or adding selected organic co-solvents. As a result, we are able to fine-tune the β-N-acetylhexosaminidase affinity and selectivity, resulting in a high-yield production of the functionalized GalNAcβ4GlcNAc disaccharide, a selective ligand of galectin-3.
Thes ynthesis of oligosaccharidesu sing mutant glycosidases hasb een dynamically developing due to the need for novelc arbohydrate-based materials.C hitooligomers (b-1!4-linkedo ligomers of N-acetylglucosamine) are bioactive compounds applicablei nm any industrial andp harmacological areas;h owever, their accessibility is still ratherl ow. In this work, GH20 b-N-acetylhexosaminidase from the fungus Talaromyces flavus wase ngineered by site-directed mutagenesis to obtain three efficiently transglycosylating variants with ca. 200-timess uppressedh ydrolytic activity.T hus,w eh ave prepared the first GH20 transglycosidases.Inthe reactions cat-alyzed by these mutant b-N-acetylhexosaminidases we were able to easily prepare andi solate bothn atural and modified chitooligomers in sufficient amountsf or their complete spectral characterization and possible further application. Thep resented method for the synthesis of chitooligomers with aglycones suitablef or linkingt oo ther biological structures is simple and robust enough to be easily scaled up.
beta-N-Acetylhexosaminidases feature so-called wobbling specificity, which means that they cleave substrates both in gluco- and galacto- configurations, with the activity ratio depending on the enzyme source. Here we present the new finding that fungal beta-N-acetylhexosaminidases are able to hydrolyze and transfer 4-deoxy-N-acetylhexosaminides with high yields. This clearly demonstrates that the 4-hydroxy moiety at the substrate pyranose ring is not essential for substrate binding to the enzyme active site, which was also confirmed by molecular docking of the tested compounds into the model of the active site of beta-N-acetylhexosaminidase from Aspergillus oryzae. A set of four 4-deoxy-N-acetylhexosaminides was synthesized and screened against a panel of beta-N-acetylhexosaminidases (extracellular and intracellular) from various sources (fungal, human, animal, plant and bacterial) for hydrolysis. The results of this screening are reported here, as well as the structures of three novel 4'-deoxy-disaccharides prepared by transglycosylation reaction with high yields (52% total disaccharide fraction) using beta-N-acetylhexosaminidase from Talaromyces flavus.
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