BACKGROUND: β-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest. RESULTS: Computational methods including homology modeling and molecular dynamics simulations were applied to shad light on the structure-activity relationship in the enzyme. Primary sequence analysis revealed some variable regions able to influence difference in substrate affinity of hexosaminidases. Moreover, docking in combination with consequent molecular dynamics simulations of C-6 modified glycosides enabled us to identify the structural features required for accommodation and processing of these bulky substrates in the active site of hexosaminidase from T. flavus. To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model. CONCLUSIONS: The main variable regions in β-N-acetylhexosaminidases determining difference in modified substrate affinity are located close to the active site entrance and engage two loops. Differences in primary sequence and the spatial arrangement of these loops and their interplay with active site amino acids, reflected by interaction energies and dynamics, account for the different catalytic activity and substrate specificity of the various fungal and bacterial β-N-acetylhexosaminidases.

Department of Structure and Function of Proteins Institute of Nanobiology and Structural Biology of GCRC Academy of Sciences of the Czech Republic Zamek 136 37333 Nove Hrady Czech Republic

Faculty of Sciences University of South Bohemia in Ceske Budejovice Zamek 136 37333 Nove Hrady Czech Republic

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

Zobrazit více v PubMed

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

Crout DHG, Singh S, Swoboda BEP, Critchley P, Gibson WT. Biotransformation in carbohydrate synthesis. N-Acetylgalactosaminyl transfer on to methyl N-acetyl-β-D-glucosaminide (methyl 2-acetamido-2-deoxy-α-D-glucopyranoside) catalysed by a β-N-acetylgalactosaminidase from Aspergillus oryzae. J Chem Soc, Chem Commun. 1992;9:704–705. doi: 10.1039/C39920000704. DOI

Carmona T, Fialová P, Křen V, Ettrich R, Martínková L, Moreno-Vargas AJ, et al. Cyanodeoxy-glycosyl derivatives as substrates for enzymatic reactions. Eur J Org Chem. 2006;2006:1876–1885. doi: 10.1002/ejoc.200500755. DOI

Fialová P, Weignerová L, Rauvolfová J, Přikrylová V, Pišvejcová A, Ettrich R, et al. 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

Ogata M, Zeng X, Usui T, Uzawa H. Substrate specificity of N-acetylhexosaminidase from Aspergillus oryzae to artificial glycosyl acceptors having various substituents at the reducing ends. Carbohydr Res. 2007;342:23–30. doi: 10.1016/j.carres.2006.11.004. PubMed DOI

Uzawa H, Zeng X, Minoura N. Synthesis of 6′-sulfodisaccharides by β-N-acetylhexosaminidase-catalyzed transglycosylation. Chem Commun. 2003;1:100–101. doi: 10.1039/B209893H. PubMed DOI

Bojarová P, Slámová K, Křenek K, Gažák R, Kulik N, Ettrich R, 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, et al. 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

Plíhal O, Sklenář J, Hofbauerová K, Novák P, Man P, Pompach P, et al. Large propeptides of fungal β-N-acetylhexosaminidases are novel enzyme regulators that must be intracellularly processed to control activity, dimerization, and secretion into the extracellular environment. Biochemistry. 2007;46:2719–2734. doi: 10.1021/bi061828m. PubMed DOI

Plíhal O, Sklenář J, Kmoníčková J, Man P, Pompach P, Havlíček V, et al. N-Glycosylated catalytic unit meets O-glycosylated propeptide: complex protein architecture in a fungal hexosaminidase. Biochem Soc Trans. 2004;32:764–765. doi: 10.1042/BST0320764. PubMed DOI

Vaněk O, Brynda J, Hofbauerová K, Kukačka Z, Pachl P, Bezouška K, et al. Crystallization and diffraction analysis of β-N-acetylhexosaminidase from Aspergillus oryzae. Acta Cryst. 2011;F67:498–503. PubMed PMC

Ettrich R, Kopecký V, Jr, Hofbauerová K, Baumruk V, Novák P, Pompach P, 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, et al. Enzymatic characterization and molecular modeling of an evolutionary interesting fungal β-N-acetylhexosaminidase. FEBS J. 2011;278:2469–2484. doi: 10.1111/j.1742-4658.2011.08173.x. PubMed DOI

Mark BL, Vocadlo DJ, Zhao D, Knapp S, Withers SG, James MN. Biochemical and structural assessment of the 1-N-azasugar GalNAc-isofagomine as a potent family 20 β-N-acetylhexosaminidase inhibitor. J Biol Chem. 2001;276:42131–42137. doi: 10.1074/jbc.M107154200. PubMed DOI

Sumida T, Ishii R, Yanagisawa T, Yokoyama S, Ito M. Molecular cloning and crystal structural analysis of a novel β-N-acetylhexosaminidase from Paenibacillus sp. TS12 capable of degrading glycosphingolipids. J Mol Biol. 2009;392:87–99. doi: 10.1016/j.jmb.2009.06.025. PubMed DOI

Jiang YL, Yu WL, Zhang JW, Frolet C, Di Guilmi AM, Zhou CZ, et al. Structural basis for the substrate specificity of a novel β-N-acetylhexosaminidase StrH protein from Streptococcus pneumoniae R6. J Biol Chem. 2011;286:43004–43012. doi: 10.1074/jbc.M111.256578. PubMed DOI PMC

Langley DB, Harty DW, Jacques NA, Hunter N, Guss JM, Collyer CA. Structure of N-acetyl-β-D-glucosaminidase (GcnA) from the endocarditis pathogen Streptococcus gordonii and its complex with the mechanism-based inhibitor NAG-thiazoline. J Mol Biol. 2008;377:104–116. doi: 10.1016/j.jmb.2007.09.028. PubMed DOI

Tews I, Perrakis A, Oppenheim A, Dauter Z, Wilson KS, Vorgias CE. Bacterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay-Sachs disease. Nature Struct Biol. 1996;3:638–648. doi: 10.1038/nsb0796-638. PubMed DOI

Ramasubbu N, Thomas LM, Ragunath C, Kaplan JB. Structural analysis of dispersin B, a biofilm-releasing glycoside hydrolase from the periodontopathogen Actinobacillus actinomycetemcomitans. J Mol Biol. 2005;349:475–486. doi: 10.1016/j.jmb.2005.03.082. PubMed DOI

Mark BL, Mahuran DJ, Cherney MM, Zhao D, Knapp S, James MN. Crystal structure of human β-hexosaminidase B: understanding the molecular basis of Sandhoff and Tay-Sachs disease. J Mol Biol. 2003;327:1093–1109. doi: 10.1016/S0022-2836(03)00216-X. PubMed DOI PMC

Lemieux MJ, Mark BL, Cherney MM, Withers SG, Mahuran DJ, James MN. Crystallographic structure of human β-hexosaminidase A: interpretation of Tay-Sachs mutations and loss of GM2 ganglioside hydrolysis. J Mol Biol. 2006;359:913–929. doi: 10.1016/j.jmb.2006.04.004. PubMed DOI PMC

Maier T, Strater N, Schuette CG, Klingenstein R, Sandhoff K, Saenger W. The X-ray crystal structure of human β-hexosaminidase B provides new insights into Sandhoff disease. J Mol Biol. 2003;328:669–681. doi: 10.1016/S0022-2836(03)00311-5. PubMed DOI

Bateman KS, Cherney MM, Mahuran DJ, Tropak M, James MN. Crystal structure of β-hexosaminidase B in complex with pyrimethamine, a potential pharmacological chaperone. J Med Chem. 2011;54(5):1421–1429. doi: 10.1021/jm101443u. PubMed DOI PMC

Liu T, Zhang H, Liu F, Wu Q, Shen X, Yang Q. Structural determinants of an insect β-N-acetyl-D-hexosaminidase specialized as a chitinolytic enzyme. J Biol Chem. 2011;286:4049–4058. doi: 10.1074/jbc.M110.184796. PubMed DOI PMC

Liu T, Zhang H, Liu F, Chen L, Shen X, Yang Q. Active-pocket size differentiating insectile from bacterial chitinolytic β-N-acetyl-D-hexosaminidases. Biochem J. 2011;438:467–474. doi: 10.1042/BJ20110390. PubMed DOI

Tews I, Terwisscha van Scheltinga AC, Perrakis A, Wilson KS, Dijkstra BW. Substrate-assisted catalysis unifies two families of chitinolytic enzymes. J Am Chem Soc. 1997;119:7954–7959. doi: 10.1021/ja970674i. DOI

Mark BL, Vocadlo DJ, Knapp S, Triggs-Raine BL, Withers SG, James MN. Crystallographic evidence for substrate-assisted catalysis in a bacterial β-hexosaminidase. J Biol Chem. 2001;276:10330–10337. doi: 10.1074/jbc.M011067200. PubMed DOI

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

Kulik N, Slámová K. Computational modelling of catalytic properties and modified substrates of fungal β-N-acetylhexosaminidases. Mini-Rev Org Chem. 2011;8:270–280. doi: 10.2174/157019311796197418. DOI

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. doi: 10.1016/S0022-2836(05)80360-2. PubMed DOI

Sali A, Blundell TL. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol. 1993;234:779–815. doi: 10.1006/jmbi.1993.1626. PubMed DOI

Davis IW, Leaver-Fay A, Chen VB, Block JN, Kapral GJ, Wang X, et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 2007;35:W375–W383. doi: 10.1093/nar/gkm216. PubMed DOI PMC

Willard L, Ranjan A, Zhang H, Monzavi H, Boyko RF, Sykes BD, et al. VADAR: a web server for quantitative evaluation of protein structure quality. Nucleic Acids Res. 2003;31:3316–3319. doi: 10.1093/nar/gkg565. PubMed DOI PMC

Bohne-Lang A, von der Lieth CW. GlyProt: in silico glycosylation of proteins. Nucleic Acids Res. 2005;33:214–219. doi: 10.1093/nar/gki385. PubMed DOI PMC

Pace CN, Shirley BA, McNutt M, Gajiwala K. Force contribution to the conformational stability of protein. FASEB J. 1996;10:75–83. PubMed

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

Liu T, Zhou Y, Chen L, Chen W, Liu L, Shen X, et al. Structural insights into cellulolytic and chitinolytic enzymes revealing crucial residues of insect β-N-acetyl-D-hexosaminidase. PLoS One. 2012;7(12):e52225. doi: 10.1371/journal.pone.0052225. PubMed DOI PMC

Notredame C, Higgins DG, Heringa J. T-coffee: a novel method for fast and accurate multiple sequence alignment. J Mol Biol. 2000;302:205–217. doi: 10.1006/jmbi.2000.4042. PubMed DOI

Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. Jalview Version 2 - a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25:1189–1191. doi: 10.1093/bioinformatics/btp033. PubMed DOI PMC

Gascuel O. BIONJ: an improved version of the NJ algorithm based on a simple model of sequence data. Mol Biol Evol. 1997;14:685–695. doi: 10.1093/oxfordjournals.molbev.a025808. PubMed DOI

Chevenet F, Brun C, Banuls AL, Jacq B, Chisten R. TreeDyn: towards dynamic graphics and annotations for analyses of trees. BMC Bioinformatics. 2006;7:439–448. doi: 10.1186/1471-2105-7-439. PubMed DOI PMC

Bernstein FC, Koetzle TF, Williams GJB, Meyer EF, Brice MD, Rodgers JR, et al. The protein data bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977;80:319–324. PubMed

Combet C, Blanchet C, Geourjon C, Deléage G. NPS@: network protein sequence analysis. Trends Biochem Sci. 2000;25:147–150. doi: 10.1016/S0968-0004(99)01540-6. PubMed DOI

Konagurthu AS, Whisstock JC, Stuckey PJ, Lesk AM. MUSTANG: a multiple structural alignment algorithm. Proteins. 2006;64:559–574. doi: 10.1002/prot.20921. PubMed DOI

Wiederstein M, Sippl MJ. ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res. 2007;35:407–410. doi: 10.1093/nar/gkm290. PubMed DOI PMC

Essman U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG. A smooth particle mesh Ewald method. J Chem Phys. 1995;103:8577–8593. doi: 10.1063/1.470117. DOI

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

Williams SJ, Mark BL, Vocadlo DJ, James MN, Withers SG. Aspartate 313 in the Streptomyces plicatus hexosaminidase plays a critical role in substrate-assisted catalysis by orienting the 2-acetamido group and stabilizing the transition state. J Biol Chem. 2002;277:40055–40065. doi: 10.1074/jbc.M206481200. PubMed DOI

Jakalian A, Jack DB, Bayly CI. 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

Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, et al. Automated docking using a Lamarckian genetic algorithm and empirical binding free energy function. J Comput Chem. 1998;19:1639–1662. doi: 10.1002/(SICI)1096-987X(19981115)19:14<1639::AID-JCC10>3.0.CO;2-B. DOI

Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, et al. Autodock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem. 2009;16:2785–2791. doi: 10.1002/jcc.21256. PubMed DOI PMC

Laskowski RA, Swindells MB. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J Chem Inf Model. 2011;51:2778–2786. doi: 10.1021/ci200227u. PubMed DOI

Mutation Hotspot for Changing the Substrate Specificity of β-N-Acetylhexosaminidase: A Library of GlcNAcases

Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation

The β-N-Acetylhexosaminidase in the Synthesis of Bioactive Glycans: Protein and Reaction Engineering