Mutation Hotspot for Changing the Substrate Specificity of β-N-Acetylhexosaminidase: A Library of GlcNAcases
Jazyk angličtina Země Švýcarsko Médium electronic
Typ dokumentu časopisecké články
Grantová podpora
22-00262S
Czech Science Foundation
LTC20072
Ministry of Education Youth and Sports
PubMed
36293310
PubMed Central
PMC9604439
DOI
10.3390/ijms232012456
PII: ijms232012456
Knihovny.cz E-zdroje
- Klíčová slova
- Pichia pastoris, Talaromyces flavus, site-directed mutagenesis, site-saturation mutagenesis, substrate specificity, β-N-acetylhexosaminidase,
- MeSH
- acetylgalaktosamin metabolismus MeSH
- acetylglukosamin * metabolismus MeSH
- acetylglukosaminidasa MeSH
- beta-N-acetylhexosaminidasy * metabolismus MeSH
- kinetika MeSH
- mutace MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- acetylgalaktosamin MeSH
- acetylglukosamin * MeSH
- acetylglukosaminidasa MeSH
- beta-N-acetylhexosaminidasy * MeSH
β-N-Acetylhexosaminidase from Talaromyces flavus (TfHex; EC 3.2.1.52) is an exo-glycosidase with dual activity for cleaving N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from carbohydrates. By targeting a mutation hotspot of the active site residue Glu332, we prepared a library of ten mutant variants with their substrate specificity significantly shifted towards GlcNAcase activity. Suitable mutations were identified by in silico methods. We optimized a microtiter plate screening method in the yeast Pichia pastoris expression system, which is required for the correct folding of tetrameric fungal β-N-acetylhexosaminidases. While the wild-type TfHex is promiscuous with its GalNAcase/GlcNAcase activity ratio of 1.2, the best single mutant variant Glu332His featured an 8-fold increase in selectivity toward GlcNAc compared with the wild-type. Several prepared variants, in particular Glu332Thr TfHex, had significantly stronger transglycosylation capabilities than the wild-type, affording longer chitooligomers - they behaved like transglycosidases. This study demonstrates the potential of mutagenesis to alter the substrate specificity of glycosidases.
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