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Flavonol 2,4-dioxygenase from Aspergillus niger DSM 821, a type 2 CuII-containing glycoprotein
H K Hund1, J Breuer, F Lingens
1Institut für Mikrobiologie, Universität Hohenheim, Stuttgart, Germany.
European Journal of Biochemistry
|September 1, 1999
Summary
Flavonol 2,4-dioxygenase from Aspergillus niger was purified and characterized as a copper-containing glycoprotein. This enzyme, crucial for rutin degradation, exhibits a unique LMS subunit structure and a distorted square planar copper-binding site.
Area of Science:
- Biochemistry
- Enzymology
- Microbial Biotechnology
Background:
- Flavonol 2,4-dioxygenase catalyzes the cleavage of quercetin.
- Understanding its structure and function is key to microbial degradation pathways.
Purpose of the Study:
- To purify and characterize the flavonol 2,4-dioxygenase enzyme from Aspergillus niger.
- To elucidate the enzyme's subunit composition, molecular mass, and metal cofactor properties.
Main Methods:
- Enzyme purification from Aspergillus niger culture filtrate.
- SDS-PAGE and mass spectrometry (MALDI-TOF-MS) for molecular mass determination.
- EPR spectroscopy to analyze the copper cofactor environment.
Main Results:
- Purified enzyme is a glycoprotein with N-linked glycan chains, molecular mass of 130-170 kDa.
- Dissociates into L (63-67 kDa), M (53-57 kDa), and S (31-35 kDa) peptides in a 1:1:1 molar ratio.
- Contains 1.0-1.6 mol copper per mol of enzyme, with EPR spectra characteristic of a type 2 Cu(II) protein in a distorted square planar geometry.
Conclusions:
- Flavonol 2,4-dioxygenase possesses a heterotrimeric LMS structure.
- The enzyme is a copper-dependent dioxygenase with a type 2 copper center.
- Characterization provides insights into the catalytic mechanism and structural properties of this important enzyme.