Open Access Highly Accessed Research article

Structure of the dimeric N-glycosylated form of fungal β-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies

Rüdiger Ettrich1*, Vladimír Kopecký2, Kateřina Hofbauerová23, Vladimír Baumruk2, Petr Novák3, Petr Pompach34, Petr Man3, Ondřej Plíhal3, Michal Kutý1, Natallia Kulik1, Jan Sklenář34, Helena Ryšlavá4, Vladimír Křen3 and Karel Bezouška34*

Author Affiliations

1 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, CZ-37333 Nové Hrady, Czech Republic

2 Institute of Physics, Faculty of Mathematics and Physics, Charles University, Ke Karlovu5, CZ-12116 Prague2, Czech Republic

3 Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská1083, CZ-14220 Prague4, Czech Republic

4 Department of Biochemistry, Faculty of Science, Charles University, Albertov2030, CZ-12840 Prague2, Czech Republic

For all author emails, please log on.

BMC Structural Biology 2007, 7:32  doi:10.1186/1472-6807-7-32

Published: 17 May 2007



Fungal β-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 β-N-acetylhexosaminidase. The fungal β-N-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from Aspergillus oryzae was purified and its sequence was determined.


The complete primary structure of the fungal β-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.


Whereas the intracellular bacterial β-N-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal β-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.