title: Computational study of β-N-acetylhexosaminidase from Talaromyces flavus, a glycosidase with high substrate flexibility.
abstract: 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.
authors: 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. kulik@nh.cas.cz.
authors: Natallia
authors: N
authors: Kulik
authors: Laboratory of Biotransformation, Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, 14220, Praha 4, Czech Republic. slamova.kristyna@gmail.com.
authors: Kristýna
authors: K
authors: Slámová
authors: 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. ettrich@nh.cas.cz.|Faculty of Sciences, University of South Bohemia in Ceske Budejovice, Zamek 136, 37333, Nove Hrady, Czech Republic. ettrich@nh.cas.cz.
authors: Rüdiger
authors: R
authors: Ettrich
authors: Laboratory of Biotransformation, Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnska 1083, 14220, Praha 4, Czech Republic. kren@biomed.cas.cz.
authors: Vladimír
authors: V
authors: Křen
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D019295:Computational Biology; D006031:Glycosylation; D007700:Kinetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D010802:Phylogeny; D015203:Reproducibility of Results; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D032901:Talaromyces / Q000201:enzymology*; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D001619:beta-N-Acetylhexosaminidases
references: J Mol Biol. 2008 Mar 14;377(1):104-16
references: 18237743
references: J Med Chem. 2011 Mar 10;54(5):1421-9
references: 21265544
references: J Mol Biol. 2005 Jun 10;349(3):475-86
references: 15878175
references: Bioinformatics. 2009 May 1;25(9):1189-91
references: 19151095
references: J Mol Biol. 2000 Sep 8;302(1):205-17
references: 10964570
references: J Biol Chem. 2011 Feb 11;286(6):4049-58
references: 21106526
references: Proteins. 2002 May 15;47(3):393-402
references: 11948792
references: Biotechnol Adv. 2010 Nov-Dec;28(6):682-93
references: 20438826
references: J Comput Chem. 2002 Dec;23(16):1623-41
references: 12395429
references: J Mol Biol. 2003 May 2;328(3):669-81
references: 12706724
references: Nucleic Acids Res. 2003 Jul 1;31(13):3316-9
references: 12824316
references: J Biol Chem. 2002 Oct 18;277(42):40055-65
references: 12171933
references: J Comput Chem. 2009 Dec;30(16):2785-91
references: 19399780
references: Biochem Soc Trans. 2004 Nov;32(Pt 5):764-5
references: 15494009
references: Trends Biochem Sci. 2000 Mar;25(3):147-50
references: 10694887
references: Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Apr 1;67(Pt 4):498-503
references: 21505251
references: Carbohydr Res. 2007 Jan 15;342(1):23-30
references: 17145046
references: Mol Biol Evol. 1997 Jul;14(7):685-95
references: 9254330
references: PLoS One. 2012;7(12):e52225
references: 23300622
references: Nucleic Acids Res. 2007 Jul;35(Web Server issue):W375-83
references: 17452350
references: J Mol Biol. 2009 Sep 11;392(1):87-99
references: 19524595
references: Biochemistry. 2007 Mar 13;46(10):2719-34
references: 17302431
references: Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W214-9
references: 15980456
references: Nat Struct Biol. 1996 Jul;3(7):638-48
references: 8673609
references: Biochem J. 2011 Sep 15;438(3):467-74
references: 21692744
references: J Mol Biol. 1990 Oct 5;215(3):403-10
references: 2231712
references: Chem Commun (Camb). 2003 Jan 7;(1):100-1
references: 12610985
references: J Biol Chem. 2011 Dec 16;286(50):43004-12
references: 22013074
references: J Mol Biol. 2003 Apr 11;327(5):1093-109
references: 12662933
references: Nucleic Acids Res. 2007 Jul;35(Web Server issue):W407-10
references: 17517781
references: BMC Bioinformatics. 2006;7:439
references: 17032440
references: J Biol Chem. 2001 Mar 30;276(13):10330-7
references: 11124970
references: Proteins. 2006 Aug 15;64(3):559-74
references: 16736488
references: FEBS J. 2011 Jul;278(14):2469-84
references: 21564548
references: BMC Struct Biol. 2007;7:32
references: 17509134
references: Protein Expr Purif. 2012 Mar;82(1):212-7
references: 22266368
references: J Biol Chem. 2001 Nov 9;276(45):42131-7
references: 11522797
references: FASEB J. 1996 Jan;10(1):75-83
references: 8566551
references: Glycobiology. 2010 Aug;20(8):1002-9
references: 20466648
references: Eur J Biochem. 1977 Nov 1;80(2):319-24
references: 923582
references: J Mol Biol. 2006 Jun 16;359(4):913-29
references: 16698036
references: J Chem Inf Model. 2011 Oct 24;51(10):2778-86
references: 21919503
references: Carbohydr Res. 2003 Apr 22;338(9):1003-8
references: 12681926
references: J Mol Biol. 1993 Dec 5;234(3):779-815
references: 8254673