Structural assembly of β-N-acetylhexosaminidase complex


Z. Kukacka1,2, P. Pompach1,2, P. Man1,2, K. Bezouska1,2 and P. Novak1,2


1Institute of Microbiology, Academy of Sciences of  the Czech Republic, 142 00 Praha

2Department of Biochemistry, Faculty of Science, Charles University in Prague, 128 00 Praha


β-N-acetylhexosaminidase (EC belongs to exoglycosidase and is one of the most abundant enzymes found in organism from bacteria to human. The fungal β‑N‑acetylhexosaminidase from Aspergillus oryzae is composed of  propeptide and catalytical domain. The propeptide is a 10kDa large peptide noncovalently associated  with the catalytical domain of the enzyme. Propeptide is essential for the enzyme activity. Although the structure of the catalytical domain was revealed by homology modeling, the structure of the propeptide has not been solved. In this study we uncover the position where the propeptide is associated with the catalytical domain.

β-N-acetylhexosaminidase was purified from the medium of the producing organism. For EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide) experiment the enzyme was transferred by gel filtration to 50mM Pyridine pH 5.5, 150mM NaCl and for DSG (disuccinimidyl glutarate) experiment to 50mM Triethylamine carbonate pH 7.5. After the cross-linking reaction was over, the products of enzyme were separated by SDS electrophoresis.  In gel digestion was performed and the resulting peptides were analyzed by LC-ESI FT MS (Agilent 1200, APEX-Ultra)

Combinating chemical cross-linking and high resolution mass spectrometry we revealed that the structural changes of the catalytical domain depend on the presence/absence of the propeptide molecule. These results nicely correlated with the previously described homology model of the catalytical domain. Several cross-linked peptides between the propeptide and catalytical domain disclosed the position of the propeptide within the enzyme.

This work was supported by grants P207/10/1040 and P207/10/1934 of the Czech Science Foundation.