The ß-N-Acetylhexosaminidase in the Synthesis of Bioactive Glycans: Protein and Reaction Engineering.
Molecules
; 24(3)2019 Feb 08.
Article
em En
| MEDLINE
| ID: mdl-30743988
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 ß-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. ß-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 ß-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 ß-N-acetylhexosaminidase affinity and selectivity, resulting in a high-yield production of the functionalized GalNAcß4GlcNAc disaccharide, a selective ligand of galectin-3.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Polissacarídeos
/
Beta-N-Acetil-Hexosaminidases
Limite:
Humans
Idioma:
En
Revista:
Molecules
Assunto da revista:
BIOLOGIA
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
República Tcheca