Volume 16, Issue 11 e202400028
Research Article

Expanding Rutinosidase Versatility: Acylated Quercetin Glucopyranosides as Substrates

Katerina Brodsky

Katerina Brodsky

Institute of Microbiology of the, Czech Academy of Sciences, Vídeňská 1083, CZ 142 00 Prague 4, Czech Republic

Department of Biochemistry and Microbiology, University of Chemistry and Technology in Prague, Technická 3, CZ 16628 Prague 6, Czech Republic

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Dr. Lucie Petrásková

Dr. Lucie Petrásková

Institute of Microbiology of the, Czech Academy of Sciences, Vídeňská 1083, CZ 142 00 Prague 4, Czech Republic

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Dr. Michal Kutý

Dr. Michal Kutý

Department of Chemistry, Faculty of Science, University of South Bohemia, Branišovská 1760, CZ 37005 České Budějovice, Czech Republic

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Assoc. Prof. Pavla Bojarová

Assoc. Prof. Pavla Bojarová

Institute of Microbiology of the, Czech Academy of Sciences, Vídeňská 1083, CZ 142 00 Prague 4, Czech Republic

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Dr. Helena Pelantová

Dr. Helena Pelantová

Institute of Microbiology of the, Czech Academy of Sciences, Vídeňská 1083, CZ 142 00 Prague 4, Czech Republic

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Prof. Vladimír Křen

Corresponding Author

Prof. Vladimír Křen

Institute of Microbiology of the, Czech Academy of Sciences, Vídeňská 1083, CZ 142 00 Prague 4, Czech Republic

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First published: 15 February 2024

Graphical Abstract

Aspergillus niger rutinosidase (AnRut) efficiently cleaved a library of rutin glycomimetics - isoquercitrin acylated at C-6′ of its glucosyl moiety. The substrates tested included isoquercitrin substituted at glucosyl C-6′ with acetyl, benzoyl, phenylacetyl, phenylpropanoyl, cinnamyl, vanillyl, galloyl, 4-hydroxybenzoyl, and 3-(4-hydroxy-3-O-methylphenyl)propanoyl. AnRut showed the ability to transglycosylate with the substrates 6′-O-acetyl isoquercitrin and 6′-O-benzoyl isoquercitrin substrates, affording n-butyl 6-acetyl-β-d-glucopyranoside and n-butyl 6-benzoyl-β-d-glucopyranoside.

Abstract

Rutinosidase is a diglycosidase that catalyzes the cleavage of rutinose (α-l-Rhap-(1→6)-β-d-Glcp) from rutin or other rutinosides. It is also able to cleave β-glucopyranosides, e. g., isoquercitrin. This enzyme has a strong transglycosylation activity and a remarkable substrate specificity. We have shown that rutinosidase from Aspergillus niger (AnRut) is able to cleave β-glucopyranosides acylated at C-6 of glucose (6′-O-acylisoquercitrin) with acetyl, benzoyl, phenylacetyl, phenylpropanoyl, cinnamoyl, vanillyl, galloyl, 4-hydroxybenzoyl and 3-(4-hydroxy-3-methoxyphenyl)propanoyl. The release of the respective 6-acylglucopyranoses was confirmed by HPLC/MS and NMR methods. Selected compounds, i. e., 6′-O-acetyl, 6′-O-benzoyl, and 6′-O-cinnamyl derivatives of isoquercitrin, were also tested as transglycosylation substrates. Only 6′-acetylisoquercitrin and 6′-O-benzoylisoquercitrin underwent transglycosylations by AnRut to produce n-butyl 6-acetyl-β-d-glucopyranoside and n-butyl 6-benzoyl-β-d-glucopyranoside. Isoquercitrin 6′-O-cinnamate yielded on hydrolytic product. Molecular modeling based on the crystal structure of AnRut showed that large aromatic moieties at C-6′ of isoquercitrin block the side tunnel of AnRut leading into its active site and thus hinder the entry of the acceptor substrate for transglycosylation. This study demonstrates the great substrate flexibility of rutinosidase at the glycone site.

Conflict of interests

The authors declare no conflict of interest.

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.