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

Pavla Bojarová,Helena Pelantová,Natalia Kulik,Michaela Hovorková,Kristýna Slámová,Vladimír Křen

doi:10.3390/molecules24030599

Pavla Bojarová, Helena Pelantová + Show 4 more

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https://doi.org/10.3390/molecules24030599

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Abstract

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.

Highlights

Introduction3.2.1.52) have the advantage of tolerating a number of functional groups incorporated in both the donor and acceptor carbohydrate molecule [3,4,5]
N-Acetylhexosamine oligosaccharides are attractive synthetic targets since they act as potent ligands of biotechnologically or biomedically attractive lectins [1,2]. β-N-Acetylhexosaminidases (EC3.2.1.52) have the advantage of tolerating a number of functional groups incorporated in both the donor and acceptor carbohydrate molecule [3,4,5]
We have recently discovered that a point mutation of the active-site amino acid Tyr to other amino acid residues, especially Phe, His, and Asn, can strongly suppress the hydrolytic activity of β-N-acetylhexosaminidases and favorably shift the reaction equilibrium towards the synthetic

Summary

Introduction

3.2.1.52) have the advantage of tolerating a number of functional groups incorporated in both the donor and acceptor carbohydrate molecule [3,4,5] Besides their natural hydrolytic activity, they can act in the synthetic (transglycosylation) mode, performing glycosylations of a range of acceptors [6,7]. We demonstrate the utility of this approach on the β-N-acetylhexosaminidase from Talaromyces flavus (Tf Hex), recombinantly expressed in Pichia pastoris [13]. This enzyme has exhibited an exceptionally broad substrate specificity and synthetic utility, even as a wild type [14]. By combining the protein and reaction engineering approaches, we were able to prepare a functionalized GalNAcβ4GlcNAc disaccharide in an over-100-mg yield from a chemically functionalized acceptor in a single reaction step

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