Rutinosidases (α-l-rhamnopyranosyl-(1-6)-β-d-glucopyranosidases, EC 3.2.1.168, CAZy GH5) are diglycosidases that cleave the glycosidic bond between the disaccharide rutinose and the respective aglycone. Similar to many retaining glycosidases, rutinosidases can also transfer the rutinosyl moiety onto acceptors with a free -OH group (so-called transglycosylation). The recombinant rutinosidase from Aspergillus niger (AnRut) is selectively produced in Pichia pastoris. It can catalyze transglycosylation reactions as an unpurified preparation directly from cultivation. This enzyme exhibits catalytic activity towards two substrates; in addition to rutinosidase activity, it also exhibits β-d-glucopyranosidase activity. As a result, new compounds are formed by β-glucosylation or rutinosylation of acceptors such as alcohols or strong inorganic nucleophiles (NaN3). Transglycosylation products with aliphatic aglycones are resistant towards cleavage by rutinosidase, therefore, their side hydrolysis does not occur, allowing higher transglycosylation yields. Fourteen compounds were synthesized by glucosylation or rutinosylation of selected acceptors. The products were isolated and structurally characterized. Interactions between the transglycosylation products and the recombinant AnRut were analyzed by molecular modeling. We revealed the role of a substrate tunnel in the structure of AnRut, which explained the unusual catalytic properties of this glycosidase and its specific transglycosylation potential. AnRut is attractive for biosynthetic applications, especially for the use of inexpensive substrates (rutin and isoquercitrin).

Center for Nanobiology and Structural Biology Institute of Microbiology of the Czech Academy of Sciences Zámek 136 CZ 37333 Nové Hrady Czech Republic

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

Institute of Biotechnology Slovak University of Technology Radlinského 9 SK 81237 Bratislava Slovakia

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

Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 CZ 14220 Prague 4 Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo nám 2 CZ 16610 Prague 6 Czech Republic

Zobrazit více v PubMed

Minig M., Mazzaferro L.S., Erra-Balsells R., Petroselli G., Breccia J.D. α-Rhamnosyl-β-glucosidase-catalyzed reactions for analysis and biotransformations of plant-based foods. J. Agric. Food Chem. 2011;59:11238–11243. doi: 10.1021/jf202412e. PubMed DOI

Gunata Z., Blondeel C., Vallier M.J., Lepoutre J.P., Sapis J.C., Watanabe N. An endoglycosidase from grape berry skin of cv. M. Alexandria hydrolyzing potentially aromatic disaccharide glycosides. J. Agric. Food Chem. 1998;46:2748–2753. doi: 10.1021/jf980084j. DOI

Neher B.D., Mazzaferro L.S., Kotik M., Oyhenart J., Halada P., Křen V., Breccia J.D. Bacteria as source of diglycosidase activity: Actinoplanes missouriensis produces 6-O-α-l-rhamnosyl-β-d-glucosidase active on flavonoids. Appl. Microbiol. Biotechnol. 2016;100:3061–3070. doi: 10.1007/s00253-015-7088-x. PubMed DOI

Weiz G., Mazzaferro L.S., Kotik M., Neher B.D., Halada P., Křen V., Breccia J.D. The flavonoid degrading fungus Acremonium sp. DSM 24697 produces two diglycosidases with different specificities. Appl. Microbiol. Biotechnol. 2019;103:9493–9504. doi: 10.1007/s00253-019-10180-y. PubMed DOI

Koseki T., Ishikawa M., Kawasaki M., Shiono Y. β-Diglycosidases from microorganisms as industrial biocatalysts: Biochemical characteristics and potential applications. Appl. Microbiol. Biotechnol. 2018;102:8717–8723. doi: 10.1007/s00253-018-9286-9. PubMed DOI

Li C.Y., Schluesener H. Health-promoting effects of the citrus flavanone hesperidin. Crit. Rev. Food Sci. Nutr. 2017;57:613–631. doi: 10.1080/10408398.2014.906382. PubMed DOI

Weng S.S., Mao L., Gong Y.Y., Sun T., Gu Q. Role of quercetin in protecting ARPE-19 cells against H2O2-induced injury via nuclear factor erythroid 2 like 2 pathway activation and endoplasmic reticulum stress inhibition. Mol. Med. Rep. 2017;16:3461–3468. doi: 10.3892/mmr.2017.6964. PubMed DOI

Boadi W.Y., Lo A. Effects of quercetin, kaempferol and exogenous glutathione on phospho- and total-AKT in 3T3-L1 preadipocytes. J. Diet. Suppl. 2018;15:814–826. doi: 10.1080/19390211.2017.1401572. PubMed DOI

Rezvan N., Moini A., Gorgani-Firuzjaee S., Hosseinzadeh-Attar M.J. Oral quercetin supplementation enhances adiponectin receptor transcript expression in polycystic ovary syndrome patients: A randomized placebo-controlled double-blind clinical trial. J. Diet. Suppl. 2018;19:627–633. doi: 10.22074/cellj.2018.4577. PubMed DOI PMC

Nam H.K., Hong S.H., Shin K.C., Oh D.K. Quercetin production from rutin by a thermostable β-rutinosidase from Pyrococcus furiosus. Biotechnol. Lett. 2012;34:483–489. doi: 10.1007/s10529-011-0786-2. PubMed DOI

Mazzaferro L.S., Breccia J.D. Functional and biotechnological insights into diglycosidases. J. Diet. Suppl. 2011;29:103–112. doi: 10.3109/10242422.2011.594882. DOI

Pageon H., Azouaoui A., Zucchi H., Ricois S., Tran C., Asselineau D. Potentially beneficial effects of rhamnose on skin ageing: An in vitro and in vivo study. Int. J. Cosmet. Sci. 2019;41:213–220. doi: 10.1111/ics.12523. PubMed DOI

Šimčíková D., Kotik M., Weignerová L., Halada P., Pelantová H., Adamcová K., Křen V. α-l-Rhamnosyl-β-d-glucosidase (rutinosidase) from Aspergillus niger: Characterization and synthetic potential of a novel diglycosidase. Adv. Synth. Catal. 2015;357:107–117. doi: 10.1002/adsc.201400566. DOI

Lombard V., Golaconda Ramulu H., Drula E., Coutinho P.M., Henrissat B. The carbohydrate-active enzyme database (CAZy) in 2013. Nucleic Acids Res. 2014;42:D490–D495. doi: 10.1093/nar/gkt1178. PubMed DOI PMC

Bassanini I., Kapešová J., Petrásková L., Pelantová H., Markošová K., Rebroš M., Valentová K., Kotik M., Káňová K., Bojarová P., et al. Glycosidase-catalyzed synthesis of glycosyl esters and phenolic glycosides of aromatic acids. Adv. Synth. Catal. 2019;361:2627–2637. doi: 10.1002/adsc.201900259. DOI

Bojarová P., Bruthans J., Křen V. β-N-Acetylhexosaminidases—The wizards of glycosylation. Appl. Microbiol. Biotechnol. 2019;103:7869–7881. doi: 10.1007/s00253-019-10065-0. PubMed DOI

Kapešová J., Petrásková L., Markošová K., Rebroš M., Kotik M., Bojarová P., Křen V. Bioproduction of quercetin and rutinose catalyzed by rutinosidase: Novel concept of “solid state biocatalysis”. Int. J. Mol. Sci. 2019;20:1112. doi: 10.3390/ijms20051112. PubMed DOI PMC

Pachl P., Kapešová J., Brynda J., Biedermannová L., Pelantová H., Bojarová P., Křen V., Řezáčová P., Kotik M. Rutinosidase from Aspergillus niger: Crystal structure and insight into the enzymatic activity. FEBS J. 2020;287:3315–3327. doi: 10.1111/febs.15208. PubMed DOI

Katayama S., Ohno F., Yamauchi Y., Kato M., Makabe H., Nakamura S. Enzymatic synthesis of novel phenol acid rutinosides using rutinase and their antiviral activity in vitro. J. Agric. Food Chem. 2013;61:9617–9622. doi: 10.1021/jf4021703. PubMed DOI

Houlmont J.P., Vercruysse K., Perez E., Rico-Lattes I., Bordat P., Treilhou M. Cosmetic use formulations containing pentyl rhamnoside and cetyl rhamnoside. Int. J. Cosmet. Sci. 2001;23:363–368. doi: 10.1046/j.0412-5463.2001.00111.x. PubMed DOI

Traber P.G., Chou H., Zomer E., Hong F., Klyosov A., Fiel M.I., Friedman S.L. Regression of fibrosis and reversal of cirrhosis in rats by galectin inhibitors in thioacetamide-induced liver disease. PLoS ONE. 2013;8:e75361. doi: 10.1371/journal.pone.0075361. PubMed DOI PMC

Bu L.T., Beckham G.T., Shirts M.R., Nimlos M.R., Adney W.S., Himmel M.E., Crowley M.F. Probing carbohydrate product expulsion from a processive cellulase with multiple absolute binding free energy methods. J. Biol. Chem. 2011;286:18161–18169. doi: 10.1074/jbc.M110.212076. PubMed DOI PMC

Varrot A., Frandsen T.P., von Ossowski I., Boyer V., Cottaz S., Driguez H., Schulein M., Davies G.J. Structural basis for ligand binding and processivity in cellobiohydrolase Cel6A from Humicola insolens. Structure. 2003;11:855–864. doi: 10.1016/S0969-2126(03)00124-2. PubMed DOI

Cutfield S.M., Davies G.J., Murshudov G., Anderson B.F., Moody P.C.E., Sullivan P.A., Cutfield J.F. The structure of the exo-β-(1,3)-glucanase from Candida albicans in native and bound forms: Relationship between a pocket and groove in family 5 glycosyl hydrolases. J. Mol. Biol. 1999;294:771–783. doi: 10.1006/jmbi.1999.3287. PubMed DOI

Taylor S.C., Ferguson A.D., Bergeron J.J.M., Thomas D.Y. The ER protein folding sensor UDP-glucose glycoprotein-glucosyltransferase modifies substrates distant to local changes in glycoprotein conformation. Nat. Struct. Mol. Biol. 2004;11:128–134. doi: 10.1038/nsmb715. PubMed DOI

Kotik M., Brodsky K., Halada P., Javůrková H., Pelantová H., Konvalinková D., Bojarová P., Křen V. Access to both anomers of rutinosyl azide using wild-type rutinosidase and its catalytic nucleophile mutant. 2020. Unpublished work.

Ly H.D., Withers S.G. Mutagenesis of glycosidases. Annu. Rev. Biochem. 1999;68:487–522. doi: 10.1146/annurev.biochem.68.1.487. PubMed DOI

Bojarová P., Kulik N., Slámová K., Hubálek M., Kotik M., Cvačka J., Pelantová H., Křen V. Selective β-N-acetylhexosaminidase from Aspergillus versicolor—A tool for producing bioactive carbohydrates. Appl. Microbiol. Biotechnol. 2019;103:1737–1753. doi: 10.1007/s00253-018-9534-z. PubMed DOI

Chovancová E., Pavelka A., Beneš P., Strnad O., Březovský J., Kozliková B., Gora A., Sustr V., Klvaňa M., Medek P., et al. CAVER 3.0: A tool for the analysis of transport pathways in dynamic protein structures. PLoS Comput. Biol. 2012;8:e1002708. doi: 10.1371/journal.pcbi.1002708. PubMed DOI PMC

Gouaux E., MacKinnon R. Principles of selective ion transport in channels and pumps. Science. 2005;310:1461–1465. doi: 10.1126/science.1113666. PubMed DOI

Klvaňa M., Pavlová M., Koudeláková T., Chaloupková R., Dvořák P., Prokop Z., Stsiapanava A., Kutý M., Kutá-Smatanová I., Dohnálek J., et al. Pathways and mechanisms for product release in the engineered haloalkane dehalogenases explored using classical and random acceleration molecular dynamics simulations. J. Mol. Biol. 2009;392:1339–1356. doi: 10.1016/j.jmb.2009.06.076. PubMed DOI

Zámocký M., Herzog C., Nykyri L.M., Koller F. Site-directed mutagenesis of the lower parts of the major substrate channel of yeast catalase A leads to highly increased peroxidatic activity. FEBS Lett. 1995;367:241–245. doi: 10.1016/0014-5793(95)00568-T. PubMed DOI

Wen Z., Baudry J., Berenbaum M.R., Schuler M.A. Ile115Leu mutation in the SRS1 region of an insect cytochrome P450 (CYP6B1) compromises substrate turnover via changes in a predicted product release channel. Protein Eng. Des. Sel. 2005;18:191–199. doi: 10.1093/protein/gzi023. PubMed DOI

Stourac J., Vavra O., Kokkonen P., Filipovic J., Pinto G., Brezovsky J., Damborsky J., Bednar D. Caver Web 1.0: Identification of tunnels and channels in proteins and analysis of ligand transport. Nucleic Acids Res. 2019;47:W414–W422. doi: 10.1093/nar/gkz378. PubMed DOI PMC

Patrick W.M., Nakatani Y., Cutfield S.M., Sharpe M.L., Ramsay R.J., Cutfield J.F. Carbohydrate binding sites in Candida albicans exo-β-1,3-glucanase and the role of the Phe-Phe ‘clamp’ at the active site entrance. FEBS J. 2010;277:4549–4561. doi: 10.1111/j.1742-4658.2010.07869.x. PubMed DOI

Mazzaferro L.S., Pinuel L., Erra-Balsells R., Giudicessi S.L., Breccia J.D. Transglycosylation specificity of Acremonium sp. α-rhamnosyl-β-glucosidase and its application to the synthesis of the new fluorogenic substrate 4-methylumbelliferyl-rutinoside. Carbohydr. Res. 2012;347:69–75. doi: 10.1016/j.carres.2011.11.008. PubMed DOI

Gerstorferová D., Fliedrová B., Halada P., Marhol P., Křen V., Weignerová L. Recombinant α-l-rhamnosidase from Aspergillus terreus in selective trimming of rutin. Process Biochem. 2012;47:828–835. doi: 10.1016/j.procbio.2012.02.014. DOI

Pavelka A. Ph.D Thesis. Masaryk University; Brno, Czech Republic: 2016. Bioinformatics Tools for the Analysis of Macromolecular Structures.

Brezovsky J., Babkova P., Degtjarik O., Fortova A., Gora A., Iermak I., Rezacova P., Dvorak P., Smatanova I.K., Prokop Z., et al. Engineering a de novo transport tunnel. ACS Catal. 2016;6:7597–7610. doi: 10.1021/acscatal.6b02081. DOI

Vavra O., Filipovic J., Plhak J., Bednar D., Marques S.M., Brezovsky J., Stourac J., Matyska L., Damborsky J. CaverDock: A molecular docking-based tool to analyse ligand transport through protein tunnels and channels. Bioinformatics. 2019;35:4986–4993. doi: 10.1093/bioinformatics/btz386. PubMed DOI

Trott O., Olson A.J. Autodock vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2010;31:455–461. doi: 10.1002/jcc.21334. PubMed DOI PMC

Kim S., Chen J., Cheng T.J., Gindulyte A., He J., He S.Q., Li Q.L., Shoemaker B.A., Thiessen P.A., Yu B., et al. PubChem 2019 update: Improved access to chemical data. Nucleic Acids Res. 2019;47:D1102–D1109. doi: 10.1093/nar/gky1033. PubMed DOI PMC

Pettersen E.F., Goddard T.D., Huang C.C., Couch G.S., Greenblatt D.M., Meng E.C., Ferrin T.E. UCSF chimera—A visualization system for exploratory research and analysis. J. Comput. Chem. 2004;25:1605–1612. doi: 10.1002/jcc.20084. PubMed DOI

Dassault Systèmes BIOVIA . Discovery Studio Modeling Environment, Release 2017. Dassault Systèmes; San Diego, CA, USA: 2016.

Berendsen H.J.C., Vanderspoel D., Vandrunen R. GROMACS—A message-passing parallel molecular-dynamics implementation. Comput. Phys. Commun. 1995;91:43–56. doi: 10.1016/0010-4655(95)00042-E. DOI

Lindahl E., Hess B., van der Spoel D. GROMACS 3.0: A package for molecular simulation and trajectory analysis. J. Mol. Model. 2001;7:306–317. doi: 10.1007/s008940100045. DOI

Flavonoids as Aglycones in Retaining Glycosidase-Catalyzed Reactions: Prospects for Green Chemistry

Two fungal flavonoid-specific glucosidases/rutinosidases for rutin hydrolysis and rutinoside synthesis under homogeneous and heterogeneous reaction conditions