Mutation Hotspot for Changing the Substrate Specificity of beta-N-Acetylhexosaminidase: A Library of GlcNAcases

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388971%3A_____%2F22%3A00563832" target="_blank" >RIV/61388971:_____/22:00563832 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.mdpi.com/1422-0067/23/20/12456" target="_blank" >https://www.mdpi.com/1422-0067/23/20/12456</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.3390/ijms232012456" target="_blank" >10.3390/ijms232012456</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Mutation Hotspot for Changing the Substrate Specificity of beta-N-Acetylhexosaminidase: A Library of GlcNAcases

Popis výsledku v původním jazyce

beta-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 beta-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.

Název v anglickém jazyce

Mutation Hotspot for Changing the Substrate Specificity of beta-N-Acetylhexosaminidase: A Library of GlcNAcases

Popis výsledku anglicky

beta-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 beta-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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2022

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

International Journal of Molecular Sciences

ISSN

1422-0067

e-ISSN

1422-0067

Svazek periodika

23

Číslo periodika v rámci svazku

20

Stát vydavatele periodika

CH - Švýcarská konfederace

Počet stran výsledku

17

Strana od-do

12456

Kód UT WoS článku

000872833300001

EID výsledku v databázi Scopus

2-s2.0-85140984333