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Microbial rutinosidases are part of the rutin catabolic pathway, enabling the microorganism to utilize rutin and related plant phenolic glycosides. Here, we report the first three\u2010dimensional structure of a rutinosidase determined at 1.27\u2010\u00c5 resolution. The rutinosidase from <jats:italic>Aspergillus\u00a0niger<\/jats:italic> K2 (<jats:italic>An<\/jats:italic>Rut), a member of glycoside hydrolase family GH\u20105, subfamily 23, was heterologously produced in <jats:italic>Pichia\u00a0pastoris<\/jats:italic>. The X\u2010ray structure of <jats:italic>An<\/jats:italic>Rut is represented by a distorted (\u03b2\/\u03b1)<jats:sub>8<\/jats:sub> barrel fold with its closest structural homologue being an <jats:italic>exo<\/jats:italic>\u2010\u03b2\u2010(1,3)\u2010glucanase from <jats:italic>Candida\u00a0albicans<\/jats:italic> (<jats:italic>Ca<\/jats:italic>Exg). The catalytic site is located in a deep pocket with a striking structural similarity to <jats:italic>Ca<\/jats:italic>Exg. However, the entrance to the active site of <jats:italic>An<\/jats:italic>Rut has been found to be different from that of <jats:italic>Ca<\/jats:italic>Exg \u2013 a mostly unstructured section of ~\u00a040 residues present in <jats:italic>Ca<\/jats:italic>Exg is missing in <jats:italic>An<\/jats:italic>Rut, whereas an additional loop of 13 amino acids partially covers the active site of <jats:italic>An<\/jats:italic>Rut. NMR analysis of reaction products provided clear evidence for a retaining reaction mechanism of <jats:italic>An<\/jats:italic>Rut. Unexpectedly, quercetin 3\u2010<jats:italic>O<\/jats:italic>\u2010glucoside was found to be a better substrate than rutin, and thus, <jats:italic>An<\/jats:italic>Rut cannot be considered a typical diglycosidase. Mutational analysis of conserved active site residues in combination with <jats:italic>in\u00a0silico<\/jats:italic> modeling allowed identification of essential interactions for enzyme activity and helped to reveal further details of substrate binding. The protein sequence of <jats:italic>An<\/jats:italic>Rut has been revised.<\/jats:p><\/jats:sec><jats:sec><jats:title>Databases<\/jats:title><jats:p>The nucleotide sequence of the rutinosidase\u2010encoding gene is available in the GenBank database under the accession number <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" ext-link-type=\"DDBJ\/EMBL\/GenBank\" xlink:href=\"MN393234\">MN393234<\/jats:ext-link>. Structural data are available in the PDB database under the accession number <jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"http:\/\/www.rcsb.org\/pdb\/search\/structidSearch.do?structureId=6I1A\">6I1A<\/jats:ext-link>.<\/jats:p><\/jats:sec><jats:sec><jats:title>Enzyme<\/jats:title><jats:p>\u03b1\u2010<jats:sc>l<\/jats:sc>\u2010Rhamnosyl\u2010\u03b2\u2010<jats:sc>d<\/jats:sc>\u2010glucosidase (<jats:ext-link xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"http:\/\/www.chem.qmul.ac.uk\/iubmb\/enzyme\/EC3\/2\/1\/168.html\">EC\u00a03.2.1.168<\/jats:ext-link>).<\/jats:p><\/jats:sec>","DOI":"10.1111\/febs.15208","type":"journal-article","created":{"date-parts":[[2020,1,13]],"date-time":"2020-01-13T14:20:15Z","timestamp":1578925215000},"page":"3315-3327","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":24,"title":["Rutinosidase from <i>Aspergillus niger<\/i>: crystal structure and insight 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