STRUCTURE OF THE b-N-ACETYLHEXOSAMINIDASE REVEALED BY
HOMOLOGY MODELING AND VIBRATIONAL SPECTROSCOPY
R. Ettrich1,
V. Kopecký Jr.2,3, K. Hofbauerová3,4,
V. Baumruk2, V. Křen4 and K. Bezouška3,4
1 Laboratory 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
2 Institute of Physics,
Charles University, Ke Karlovu 5, Prague 2, CZ-12116, Czech Republic
3 Department of
Biochemistry, Faculty of Science, Charles University, Albertov 2030, Prague 2,
CZ-12840, Czech Republic, hofbauer@biomed.cas.cz
4 Institute 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 and S. plicatus (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
docking of the chitobiose was explored with the DOCK module of SYBYL. 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. To test
thermostability of the enzyme, Raman spectra in the 5–60 °C temperature range
were recorded and analyzed by means of factor analysis.
Support from the Ministry of Education of the
Czech Republic (No. MSM113100001, No. MSM113200002, No. LN00A141),
from the Institutional Research Concept AVOZ5020903, from the Grant Agency of
the Czech Republic (grant No. 203/04/1045), and from the Grant Agency of the
Academy of Sciences (A5020403) 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.