STRUCTURE AND DYNAMICS OF THE b-N-ACETYLHEXOSAMINIDASE IN NATIVE AND DEGLYCOSYLED FORM REVEALED BY HOMOLOGY MODELING AND VIBRATIONAL SPECTROSCOPY
R. Ettrich,1 V. Kopecký Jr.,2
K. Hofbauerová,3,4 V. Baumruk,2 and
K. Bezouška3,4
1Laboratory of High Performance Computing,
Institute of Physical Biology of USB and Institute of Landscape Ecology of AS
CR, Zámek 136, Nové Hrady, CZ-37333, Czech Republic
2Institute of Physics, Faculty of
Mathematics and Physics, Charles University, Ke Karlovu 5, Prague 2, CZ-12116,
Czech Republic
3Department of Biochemistry, Faculty of
Science, Charles University, Albertov 2030, Prague 2, CZ-12840, Czech
Republic, e-mail: hofbauer@biomed.cas.cz
4Institute of Microbiology, Academy of
Sciences of the Czech Republic, Vídeňská 1083, Prague 4, CZ-14220, Czech
Republic
Glycoprotein b-N-acetylhexosaminidase from Aspergillus oryzae catalyses hydrolysis of chitobiose into constituent monosaccharides. The enzyme is physiologically important during the life cycle of fungi [1]. There is an interest in the catalytic mechanism by which these enzymes cleave their substrate since these processes are important in human diseases and control of fungal and insect pests.
Homology
modeling with the Modeller program [2] used the coordinates for a S. marcescens, S. plicatus and human
lysosomal-hexosaminidase (enzymes of glycohydrolase family 20 with a 44% degree
of similarity). Refinement was achieved through algorithmic analysis and
minimization with the TRIPOS force field in the SYBYL/MAXIMIN2 module. The
model structure has been confronted with data from FTIR and Raman spectroscopy
obtained with the enzyme (both native and deglycosylated form) purified from
the medium of the producing organism A.
oryzae. The secondary structure determined from analysis of FTIR amide I
and II bands and Raman amide I corresponds well to our molecular model. The
results from Raman and FTIR spectroscopy demonstrate the differences in
secondary structure content caused by deglycosylation of the protein.
Thermal dynamics in the range of 5–70 °C monitored by Raman spectroscopy and analyzed by homospectral 2D-correlation analysis revealed also significant changes in dynamics of proteins in native and deglycosyled forms. Glycans attached on the surface of the protein stabilize mostly hydrophobic parts of b-sheet structures that can prevent the proteins from aggregation. Therefore, we can conclude that our model of b-N-acetylhexosaminidase brings better understanding of the protein structure and dynamics with respect to its carbohydrate part.
Support
from the Institutional Research Concept of the Academy of Science of the Czech
Republic (No. AVOZ50200510, No. AVOZ60870520) and from the Ministry of
Education of the Czech Republic (No. MSM0021620835, No. MSM6007665808) and the
Grant Agency of the Czech Republic (No. 203/04/1045) is gratefully
acknowledged.
[1] Q. Cheng, H. Li, K. Merdek, J. T. Park, J. Bacteriol. 182 (2000) 4836–4840.
[2] A. Sali and J.P. Overington, Protein Sci. 3 (1994) 1582–1596.