β-N-Acetylhexosaminidase from Talaromyces flavus (TfHex; EC 3.2.1.52) is an exo-glycosidase with dual activity for cleaving N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from carbohydrates. By targeting a mutation hotspot of the active site residue Glu332, we prepared a library of ten mutant variants with their substrate specificity significantly shifted towards GlcNAcase activity. Suitable mutations were identified by in silico methods. We optimized a microtiter plate screening method in the yeast Pichia pastoris expression system, which is required for the correct folding of tetrameric fungal β-N-acetylhexosaminidases. While the wild-type TfHex is promiscuous with its GalNAcase/GlcNAcase activity ratio of 1.2, the best single mutant variant Glu332His featured an 8-fold increase in selectivity toward GlcNAc compared with the wild-type. Several prepared variants, in particular Glu332Thr TfHex, had significantly stronger transglycosylation capabilities than the wild-type, affording longer chitooligomers - they behaved like transglycosidases. This study demonstrates the potential of mutagenesis to alter the substrate specificity of glycosidases.

Department of Genetics and Microbiology Faculty of Science Charles University Viničná 5 CZ 12843 Praha 2 Czech Republic

Laboratory of Biotransformation Institute of Microbiology of the Czech Academy of Sciences Vídeňská 1083 CZ 14220 Praha 4 Czech Republic

Laboratory of Structural Biology and Bioinformatics Institute of Microbiology of the Czech Academy of Sciences Zámek 136 CZ 37333 Nové Hrady Czech Republic

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Muschiol J., Vuillemin M., Meyer A.S., Zeuner B. β-N-Acetylhexosaminidases for carbohydrate synthesis via trans-glycosylation. Catalysts. 2020;10:365. doi: 10.3390/catal10040365. DOI

Slámová K., Bojarová P., Petrásková L., Křen V. β-N-Acetylhexosaminidase: What’s in a name…? Biotechnol. Adv. 2010;28:682–693. doi: 10.1016/j.biotechadv.2010.04.004. PubMed DOI

Rauvolfová J., Kuzma M., Weignerová L., Fialová P., Přikrylová V., Pišvejcová A., Macková M., Křen V. β-N-Acetylhexosaminidase-catalysed synthesis of non-reducing oligosaccharides. J. Mol. Catal. B Enzym. 2004;29:233–239. doi: 10.1016/j.molcatb.2003.10.008. DOI

Fialová P., Weignerová L., Rauvolfová J., Přikrylová V., Pišvejcová A., Ettrich R., Kuzma M., Sedmera P., Křen V. Hydrolytic and transglycosylation reactions of N-acyl modified substrates catalysed by β-N-acetylhexosaminidases. Tetrahedron. 2004;60:693–701. doi: 10.1016/j.tet.2003.10.111. DOI

Slámová K., Bojarová P. Engineered N-acetylhexosamine-active enzymes in glycoscience. BBA—Gen. Subj. 2017;1861:2070–2087. doi: 10.1016/j.bbagen.2017.03.019. PubMed 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

Chen X., Jin L., Jiang X., Guo L., Gu G., Xu L., Lu L., Wang F., Xiao M. Converting a β-N-acetylhexosaminidase into two trans-β-N-acetylhexosaminidases by domain-targeted mutagenesis. Appl. Microbiol. Biotechnol. 2020;104:661–673. doi: 10.1007/s00253-019-10253-y. PubMed DOI

Jamek S.B., Muschiol J., Holck J., Zeuner B., Busk P.K., Mikkelsen J.D., Meyer A.S. Loop protein engineering for improved transglycosylation activity of a b-N-acetylhexosaminidase. ChemBioChem. 2018;19:1858–1865. doi: 10.1002/cbic.201800181. PubMed DOI

Weignerová L., Vavrušková P., Pišvejcová A., Thiem J., Křen V. Fungal β-N-acetylhexosaminidases with high β-N-acetylgalactosaminidase activity and their use for synthesis of β-GalNAc-containing oligosaccharides. Carbohydr. Res. 2003;338:1003–1008. doi: 10.1016/S0008-6215(03)00044-2. 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

Nekvasilová P., Andreasová I., Petrásková L., Pelantová H., Křen V., Bojarová P. A novel enzymatic tool for transferring GalNAc moiety onto challenging acceptors. BBA—Proteins Proteom. 2020;1868:140319. doi: 10.1016/j.bbapap.2019.140319. PubMed DOI

Chen X., Xu L., Jin L., Sun B., Gu G., Lu L., Xiao M. Efficient and regioselective synthesis of β-GalNAc/GlcNAc-lactose by a bifunctional transglycosylating β-N-acetylhexosaminidase from Bifidobacterium bifidum. Appl. Environ. Microbiol. 2016;82:5642. doi: 10.1128/AEM.01325-16. PubMed DOI PMC

Kurakake M., Goto T., Ashiki K., Suenaga Y., Komaki T. Synthesis of new glycosides by transglycosylation of N-acetylhexosaminidase from Serratia marcescens YS-1. J. Agric. Food Chem. 2003;51:1701–1705. doi: 10.1021/jf020965x. PubMed DOI

Visnapuu T., Teze D., Kjeldsen C., Lie A., Duus J.Ø., André-Miral C., Pedersen L.H., Stougaard P., Svensson B. Identification and characterization of a β-N-acetylhexosaminidase with a biosynthetic activity from the marine bacterium Paraglaciecola hydrolytica S66T. Int. J. Mol. Sci. 2020;21:417. doi: 10.3390/ijms21020417. PubMed DOI PMC

Roth C., Petricevic M., John A., Goddard-Borger E.D., Davies G.J., Williams S.J. Structural and mechanistic insights into a Bacteroides vulgatus retaining N-acetyl-β-galactosaminidase that uses neighbouring group participation. Chem. Commun. 2016;52:11096–11099. doi: 10.1039/C6CC04649E. PubMed DOI

Noach I., Pluvinage B., Laurie C., Abe K.T., Alteen M.G., Vocadlo D.J., Boraston A.B. The Details of glycolipid glycan hydrolysis by the structural analysis of a family 123 glycoside hydrolase from Clostridium perfringens. J. Mol. Biol. 2016;428:3253–3265. doi: 10.1016/j.jmb.2016.03.020. PubMed DOI

Sumida T., Fujimoto K., Ito M. Molecular cloning and catalytic mechanism of a novel glycosphingolipid-degrading β-N-acetylgalactosaminidase from Paenibacillus sp. TS12. J. Biol. Chem. 2011;286:14065–14072. doi: 10.1074/jbc.M110.182592. PubMed DOI PMC

Gloster T.M., Vocadlo D.J. Mechanism, structure, and inhibition of O-GlcNAc processing enzymes. Curr. Signal Transduct. Ther. 2010;5:74–91. doi: 10.2174/157436210790226537. PubMed DOI PMC

Krejzová J., Šimon P., Kalachova L., Kulik N., Bojarová P., Marhol P., Pelantová H., Cvačka J., Ettrich R., Slámová K., et al. Inhibition of GlcNAc-processing glycosidases by C-6-azido-NAG-thiazoline and its derivatives. Molecules. 2014;19:3471–3488. doi: 10.3390/molecules19033471. PubMed DOI PMC

Bojarová P., Křenek K., Kuzma M., Petrásková L., Bezouška K., Namdjou D.-J., Elling L., Křen V. N-Acetylhexosamine triad in one molecule: Chemoenzymatic introduction of 2-acetamido-2-deoxy-β-d-galactopyranosyluronic acid residue into a complex oligosaccharide. J. Mol. Catal. B Enzym. 2008;50:69–73. doi: 10.1016/j.molcatb.2007.09.002. DOI

Bojarová P., Slámová K., Křenek K., Gažák R., Kulik N., Ettrich R., Pelantová H., Kuzma M., Riva S., Adámek D., et al. Charged hexosaminides as new substrates for β-N-acetylhexosaminidase-catalyzed synthesis of immunomodulatory disaccharides. Adv. Synth. Catal. 2011;353:2409–2420. doi: 10.1002/adsc.201100371. DOI

Slámová K., Gažák R., Bojarová P., Kulik N., Ettrich R., Pelantová H., Sedmera P., Křen V. 4-Deoxy-substrates for β-N-acetylhexosaminidases: How to make use of their loose specificity. Glycobiology. 2010;20:1002–1009. doi: 10.1093/glycob/cwq058. PubMed DOI

Bojarová P., Kulik N., Hovorková M., Slámová K., Pelantová H., Křen V. The β-N-acetylhexosaminidase in the synthesis of bioactive glycans: Protein and reaction engineering. Molecules. 2019;24:599. doi: 10.3390/molecules24030599. PubMed DOI PMC

Ettrich R., Kopecký V., Hofbauerová K., Baumruk V., Novák P., Pompach P., Man P., Plíhal O., Kutý M., Kulik N., et al. Structure of the dimeric N-glycosylated form of fungal β-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies. BMC Struct. Biol. 2007;7:32. doi: 10.1186/1472-6807-7-32. PubMed DOI PMC

Ryšlavá H., Kalendová A., Doubnerová V., Skočdopol P., Kumar V., Kukačka Z., Pompach P., Vaněk O., Slámová K., Bojarová P., et al. Enzymatic characterization and molecular modeling of an evolutionarily interesting fungal β-N-acetylhexosaminidase. FEBS J. 2011;278:2469–2484. doi: 10.1111/j.1742-4658.2011.08173.x. PubMed DOI

Fialová P., Carmona A.T., Robina I., Ettrich R., Sedmera P., Přikrylová V., Petrásková-Hušáková L., Křen V. Glycosyl azide—A novel substrate for enzymatic transglycosylations. Tetrahedron Lett. 2005;46:8715–8718. doi: 10.1016/j.tetlet.2005.10.040. DOI

Slámová K., Krejzová J., Marhol P., Kalachova L., Kulik N., Pelantová H., Cvačka J., Křen V. Synthesis of derivatized chitooligomers using transglycosidases engineered from the fungal GH20 β-N-acetylhexosaminidase. Adv. Synth. Catal. 2015;357:1941–1950. doi: 10.1002/adsc.201500075. DOI

Kapešová J., Petrásková L., Kulik N., Straková Z., Bojarová P., Markošová K., Rebroš M., Křen V., Slámová K. Transglycosidase activity of glycosynthase-type mutants of a fungal GH20 β-N-acetylhexosaminidase. Int. J. Biol. Macromol. 2020;161:1206–1215. doi: 10.1016/j.ijbiomac.2020.05.273. PubMed DOI

Mackenzie L.F., Wang Q., Warren R.A.J., Withers S.G. Glycosynthases:  Mutant glycosidases for oligosaccharide synthesis. J. Am. Chem. Soc. 1998;120:5583–5584. doi: 10.1021/ja980833d. DOI

Malet C., Planas A. From β-glucanase to β-glucansynthase: Glycosyl transfer to α-glycosyl fluorides catalyzed by a mutant endoglucanase lacking its catalytic nucleophile. FEBS Lett. 1998;440:208–212. doi: 10.1016/S0014-5793(98)01448-3. PubMed DOI

Mészáros Z., Petrásková L., Kulik N., Pelantová H., Bojarová P., Křen V., Slámová K. Hypertransglycosylating variants of the GH20 β-N-acetylhexosaminidase for the synthesis of chitooligomers. Adv. Synth. Catal. 2022;364:2009–2022. doi: 10.1002/adsc.202200046. DOI

Nekvasilová P., Kulik N., Rychlá N., Pelantová H., Petrásková L., Bosáková Z., Cvačka J., Slámová K., Křen V., Bojarová P. How site-directed mutagenesis boosted selectivity of a promiscuous enzyme. Adv. Synt. Catal. 2020;362:4138–4150. doi: 10.1002/adsc.202000604. DOI

Bojarová P., Chytil P., Mikulová B., Bumba L., Konefał R., Pelantová H., Krejzová J., Slámová K., Petrásková L., Kotrchová L., et al. Glycan-decorated HPMA copolymers as high-affinity lectin ligands. Polym. Chem. 2017;8:2647–2658. doi: 10.1039/C7PY00271H. DOI

Bojarová P., Tavares M.R., Laaf D., Bumba L., Petrásková L., Konefał R., Bláhová M., Pelantová H., Elling L., Etrych T., et al. Biocompatible glyconanomaterials based on HPMA-copolymer for specific targeting of galectin-3. J. Nanobiotechnol. 2018;16:73. doi: 10.1186/s12951-018-0399-1. PubMed DOI PMC

Laaf D., Bojarová P., Mikulová B., Pelantová H., Křen V., Elling L. Two-step enzymatic synthesis of β-d-N-acetylgalactosamine-(1 → 4)-d-N-acetylglucosamine (LacdiNAc) chitooligomers for deciphering galectin binding behavior. Adv. Synth. Catal. 2017;359:2101–2108. doi: 10.1002/adsc.201700331. DOI

Drozdová A., Bojarová P., Křenek K., Weignerová L., Henßen B., Elling L., Christensen H., Jensen H.H., Pelantová H., Kuzma M., et al. Enzymatic synthesis of dimeric glycomimetic ligands of NK cell activation receptors. Carbohydr. Res. 2011;346:1599–1609. doi: 10.1016/j.carres.2011.04.043. PubMed DOI

Kulik N., Slámová K., Ettrich R., Křen V. Computational study of β-N-acetylhexosaminidase from Talaromyces flavus, a glycosidase with high substrate flexibility. BMC Bioinf. 2015;16:28. doi: 10.1186/s12859-015-0465-8. PubMed DOI PMC

Škerlová J., Bláha J., Pachl P., Hofbauerová K., Kukačka Z., Man P., Pompach P., Novák P., Otwinowski Z., Brynda J., et al. Crystal structure of native b-N-acetylhexosaminidase isolated from Aspergillus oryzae sheds light onto its substrate specificity, high stability, and regulation by propeptide. FEBS J. 2018;285:580–598. doi: 10.1111/febs.14360. PubMed DOI

Slámová K., Bojarová P., Gerstorferová D., Fliedrová B., Hofmeisterová J., Fiala M., Pompach P., Křen V. Sequencing, cloning and high-yield expression of a fungal β-N-acetylhexosaminidase in Pichia pastoris. Protein Expression Purif. 2012;82:212–217. doi: 10.1016/j.pep.2012.01.004. PubMed DOI

Karbalaei M., Rezaee S.A., Farsiani H. Pichia pastoris: A highly successful expression system for optimal synthesis of heterologous proteins. J. Cell. Physiol. 2020;235:5867–5881. doi: 10.1002/jcp.29583. PubMed DOI PMC

Marx H., Mecklenbräuker A., Gasser B., Sauer M., Mattanovich D. Directed gene copy number amplification in Pichia pastoris by vector integration into the ribosomal DNA locus. FEMS Yeast Res. 2009;9:1260–1270. doi: 10.1111/j.1567-1364.2009.00561.x. PubMed DOI

Reetz M.T. Laboratory evolution of stereoselective enzymes as a means to expand the toolbox of organic chemists. Tetrahedron. 2012;68:7530–7548. doi: 10.1016/j.tet.2012.05.093. DOI

Reetz M.T., Kahakeaw D., Lohmer R. Addressing the numbers problem in directed evolution. ChemBioChem. 2008;9:1797–1804. doi: 10.1002/cbic.200800298. PubMed DOI

Weis R., Luiten R., Skranc W., Schwab H., Wubbolts M., Glieder A. Reliable high-throughput screening with Pichia pastoris by limiting yeast cell death phenomena. FEMS Yeast Res. 2004;5:179–189. doi: 10.1016/j.femsyr.2004.06.016. PubMed DOI

Mansur M., Cabello C., Hernández L., País J., Varas L., Valdés J., Terrero Y., Hidalgo A., Plana L., Besada V., et al. Multiple gene copy number enhances insulin precursor secretion in the yeast Pichia pastoris. Biotechnol. Lett. 2005;27:339–345. doi: 10.1007/s10529-005-1007-7. PubMed DOI

Vassileva A., Arora Chugh D., Swaminathan S., Khanna N. Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris. Protein Expression Purif. 2001;21:71–80. doi: 10.1006/prep.2000.1335. PubMed DOI

Chen Y., Zhang B., Pei H., Lv J., Yang W., Cao Y., Dong B. Directed evolution of Penicillium janczewskii zalesk α-galactosidase toward enhanced activity and expression in Pichia pastoris. Appl. Biochem. Biotechnol. 2012;168:638–650. doi: 10.1007/s12010-012-9806-5. PubMed DOI

Kao M.R., Yu S.M., Ho T.U.D. Improvements of the productivity and saccharification efficiency of the cellulolytic β-glucosidase D2-BGL in Pichia pastoris via directed evolution. Biotechnol. Biofuels. 2021;14:126. doi: 10.1186/s13068-021-01973-3. PubMed DOI PMC

Beard H., Cholleti A., Pearlman D., Sherman W., Loving K.A. Applying physics-based scoring to calculate free energies of binding for single amino acid mutations in protein-protein complexes. PLoS ONE. 2013;8:e82849. doi: 10.1371/journal.pone.0082849. PubMed DOI PMC

Liu T., Zhou Y., Chen L., Chen W., Liu L., Shen X., Zhang W., Zhang J., Yang Q. Structural Insights into cellulolytic and chitinolytic enzymes revealing crucial residues of insect β-N-acetyl-D-hexosaminidase. PLoS ONE. 2012;7:e52225. doi: 10.1371/journal.pone.0052225. PubMed DOI PMC

Hovorková M., Kulik N., Konvalinková D., Petrásková L., Křen V., Bojarová P. Mutagenesis of catalytic nucleophile of β-galactosidase retains residual hydrolytic activity and affords a transgalactosidase. ChemCatChem. 2021;13:4532–4542. doi: 10.1002/cctc.202101107. DOI

Ochoa R., Soler M.A., Laio A., Cossio P. Assessing the capability of in silico mutation protocols for predicting the finite temperature conformation of amino acids. Phys. Chem. Chem. Phys. 2018;20:25901–25909. doi: 10.1039/C8CP03826K. PubMed DOI

Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. PubMed DOI

Garcia-Oliva C., Hoyos P., Petrásková L., Kulik N., Pelantová H., Cabanillas A.H., Rumbero Á., Křen V., Hernáiz M.J., Bojarová P. Acceptor specificity of β-N-acetylhexosaminidase from Talaromyces flavus: A rational explanation. Int. J. Mol. Sci. 2019;20:6181. doi: 10.3390/ijms20246181. PubMed DOI PMC

Konagurthu A.S., Whisstock J.C., Stuckey P.J., Lesk A.M. MUSTANG: A multiple structural alignment algorithm. Proteins. 2006;64:559–574. doi: 10.1002/prot.20921. PubMed DOI

Krieger E., Koraimann G., Vriend G. Increasing the precision of comparative models with YASARA NOVA-a self-parameterizing force field. Proteins: Struct. Funct. Bioinf. 2002;47:393–402. doi: 10.1002/prot.10104. PubMed DOI

Schrödinger Release 2018-4: Build 12. S. LLC; New York, NY, USA: 2018.

Canutescu A.A., Shelenkov A.A., Dunbrack R.L., Jr. A graph-theory algorithm for rapid protein side-chain prediction. Protein Sci. 2003;12:2001–2014. doi: 10.1110/ps.03154503. PubMed DOI PMC

Kirschner K.N., Yongye A.B., Tschampel S.M., González-Outeiriño J., Daniels C.R., Foley B.L., Woods R.J. GLYCAM06: A generalizable biomolecular force field. Carbohydrates. J. Comput. Chem. 2008;29:622–655. doi: 10.1002/jcc.20820. PubMed DOI PMC

Jakalian A., Jack D.B., Bayly C.I. Fast, efficient generation of high-quality atomic charges. AM1-BCC model: II. Parameterization and validation. J. Comput. Chem. 2002;23:1623–1641. doi: 10.1002/jcc.10128. PubMed DOI

Krieger E., Joo K., Lee J., Lee J., Raman S., Thompson J., Tyka M., Baker D., Karplus K. Improving physical realism, stereochemistry, and side-chain accuracy in homology modeling: Four approaches that performed well in CASP8. Proteins. 2009;77((Suppl. 9)):114–122. doi: 10.1002/prot.22570. PubMed DOI PMC

Berendsen H.J.C., Postma J.P.M., van Gunsteren W.F., DiNola A., Haak J.R. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 1984;81:3684–3690. doi: 10.1063/1.448118. DOI

Land H., Humble M.S. YASARA: A Tool to Obtain Structural Guidance in Biocatalytic Investigations. In: Bornscheuer U.T., Höhne M., editors. Protein Engineering: Methods and Protocols. Springer; New York, NY, USA: 2018. pp. 43–67. PubMed

Turner P.J. XMGRACE, Version 5.1.19. Center for Coastal and Land-Margin Research, Oregon Graduate Institute of Science and Technology; Beaverton, OR, USA: 2005.