Mutagenesis of Catalytic Nucleophile of beta-Galactosidase Retains Residual Hydrolytic Activity and Affords a Transgalactosidase

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388971%3A_____%2F21%3A00549199" target="_blank" >RIV/61388971:_____/21:00549199 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216208:11310/21:10436608

Výsledek na webu

<a href="https://chemistry-europe.onlinelibrary.wiley.com/doi/epdf/10.1002/cctc.202101107" target="_blank" >https://chemistry-europe.onlinelibrary.wiley.com/doi/epdf/10.1002/cctc.202101107</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1002/cctc.202101107" target="_blank" >10.1002/cctc.202101107</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Mutagenesis of Catalytic Nucleophile of beta-Galactosidase Retains Residual Hydrolytic Activity and Affords a Transgalactosidase

Popis výsledku v původním jazyce

Glycosidases that cleave oligosaccharides can also synthesize the glycosidic bond. Site-directed mutagenesis of the catalytic nucleophile commonly abolishes their hydrolytic activity, affording glycosynthases that use glycosyl fluorides as substrates. Here, the synthetic ability of beta-galactosidase from Bacillus circulans isoform A (BgaD-A, EC 3.2.1.23, GH2) was investigated by site-directed mutagenesis. The cold-shock expression ensured selective induction and correct folding. Three mutants were constructed at the active-site catalytic nucleophile E532 as putative glycosynthases. However, none of the mutants could process alpha-galactosyl fluoride as a galactosyl donor. With only negligible hydrolytic activity, two mutants selectively synthesized azido-functionalized N-acetyllactosamine using the p-nitrophenyl beta-d-galactoside as a galactosyl donor. Thus, they behaved as transglycosidases. This study demonstrates that substitution at the catalytic nucleophile for the assembly of glycosynthases is not unrestrictedly versatile and that the effect of mutagenesis on synthetic abilities depends on the relative orientations of amino acids in the enzyme active site.

Název v anglickém jazyce

Mutagenesis of Catalytic Nucleophile of beta-Galactosidase Retains Residual Hydrolytic Activity and Affords a Transgalactosidase

Popis výsledku anglicky

Glycosidases that cleave oligosaccharides can also synthesize the glycosidic bond. Site-directed mutagenesis of the catalytic nucleophile commonly abolishes their hydrolytic activity, affording glycosynthases that use glycosyl fluorides as substrates. Here, the synthetic ability of beta-galactosidase from Bacillus circulans isoform A (BgaD-A, EC 3.2.1.23, GH2) was investigated by site-directed mutagenesis. The cold-shock expression ensured selective induction and correct folding. Three mutants were constructed at the active-site catalytic nucleophile E532 as putative glycosynthases. However, none of the mutants could process alpha-galactosyl fluoride as a galactosyl donor. With only negligible hydrolytic activity, two mutants selectively synthesized azido-functionalized N-acetyllactosamine using the p-nitrophenyl beta-d-galactoside as a galactosyl donor. Thus, they behaved as transglycosidases. This study demonstrates that substitution at the catalytic nucleophile for the assembly of glycosynthases is not unrestrictedly versatile and that the effect of mutagenesis on synthetic abilities depends on the relative orientations of amino acids in the enzyme active site.

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

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2021

Kód důvěrnosti údajů

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

Název periodika

ChemCatChem

ISSN

1867-3880

e-ISSN

1867-3899

Svazek periodika

13

Číslo periodika v rámci svazku

21

Stát vydavatele periodika

DE - Spolková republika Německo

Počet stran výsledku

11

Strana od-do

4532-4542

Kód UT WoS článku

000698251800001

EID výsledku v databázi Scopus

2-s2.0-85115295801