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

Result code in 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>
Alternative codes found
RIV/00216208:11310/21:10436608
Result on the web
<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>

Result language
angličtina
Original language name
Mutagenesis of Catalytic Nucleophile of beta-Galactosidase Retains Residual Hydrolytic Activity and Affords a Transgalactosidase
Original language description
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.
Czech name
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Czech description
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Type
J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database
CEP classification
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OECD FORD branch
10608 - Biochemistry and molecular biology

Project
Result was created during the realization of more than one project. More information in the Projects tab.
Continuities
P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Publication year
2021
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

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