title: Structure of the dimeric N-glycosylated form of fungal beta-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies.
abstract: BACKGROUND
  Fungal beta-N-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal beta-N-acetylhexosaminidase. The fungal beta-N-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from Aspergillus oryzae was purified and its sequence was determined.
  
  
  RESULTS
  The complete primary structure of the fungal beta-N-acetylhexosaminidase from Aspergillus oryzae CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the N-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate - chitobiose with a stable value of binding energy during the molecular dynamics simulation.
  
  
  CONCLUSIONS
  Whereas the intracellular bacterial beta-N-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal beta-N-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected enzyme stability in acidic conditions. Dimerization and N-glycosylation are the enzyme's strategy for catalytic subunit stabilization. The disulfide bridge that connects Cys448 with Cys483 stabilizes a hinge region in a flexible loop close to the active site, which is an exclusive feature of the fungal enzymes, neither present in bacterial nor mammalian structures. This loop may play the role of a substrate binding site lid, anchored by a disulphide bridge that prevents the substrate binding site from being influenced by the flexible motion of the loop.
authors.affiliation: Laboratory of High Performance Computing, Institute of Systems Biology and Ecology of the Academy of Sciences of the Czech Republic and Institute of Physical Biology of USB, Zámek136, Nové Hrady, Czech Republic. ettrich@greentech.cz
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Ettrich
authors.forename: Vladimír
authors.initials: V
authors.lastname: Kopecký
authors.forename: Katerina
authors.initials: K
authors.lastname: Hofbauerová
authors.forename: Vladimír
authors.initials: V
authors.lastname: Baumruk
authors.forename: Petr
authors.initials: P
authors.lastname: Novák
authors.forename: Petr
authors.initials: P
authors.lastname: Pompach
authors.forename: Petr
authors.initials: P
authors.lastname: Man
authors.forename: Ondrej
authors.initials: O
authors.lastname: Plíhal
authors.forename: Michal
authors.initials: M
authors.lastname: Kutý
authors.forename: Natallia
authors.initials: N
authors.lastname: Kulik
authors.forename: Jan
authors.initials: J
authors.lastname: Sklenár
authors.forename: Helena
authors.initials: H
authors.lastname: Ryslavá
authors.forename: Vladimír
authors.initials: V
authors.lastname: Kren
authors.forename: Karel
authors.initials: K
authors.lastname: Bezouska
mesh_terms: D001236:Aspergillus oryzae / Q000201:enzymology*; D003198:Computer Simulation; D019281:Dimerization; D004795:Enzyme Stability; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013059:Spectrum Analysis, Raman / Q000379:methods*; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001619:beta-N-Acetylhexosaminidases