Gentisate 1,2-dioxygenase from Xanthobacter polyaromaticivorans 127W
Shin-ichi Hirano1, Masaaki Morikawa, Kazufumi Takano
1Department of Material and Life Science, Graduate School of Engineering, Osaka University, Japan.
Bioscience, Biotechnology, and Biochemistry
|January 11, 2007
Summary
A novel gentisate 1,2-dioxygenase (GDOxp) from Xanthobacter polyaromaticivorans exhibits broad substrate specificity and tetrameric structure. It can reversibly inactivate and reactivate, with its N-terminus crucial for tetramerization.
Area of Science:
- Microbial metabolism
- Enzymology
- Biochemistry
Background:
- Dibenzothiophene degradation involves specific dioxygenase enzymes.
- Xanthobacter polyaromaticivorans harbors a gene cluster (dbd) for dibenzothiophene degradation.
- Dioxygenases play critical roles in the breakdown of aromatic compounds.
Purpose of the Study:
- To characterize a putative gentisate 1,2-dioxygenase (GDOxp) from Xanthobacter polyaromaticivorans.
- To investigate the substrate specificity and structural properties of GDOxp.
- To understand the factors influencing GDOxp activity and stability.
Main Methods:
- Heterologous expression of GDOxp in Escherichia coli.
- Purification of the recombinant enzyme to homogeneity.
- Enzyme activity assays with various substrates.
- Analysis of enzyme quaternary structure and stability under different conditions.
- Protein truncation and digestion experiments to identify functional regions.
Main Results:
- The GDOxp enzyme showed high sequence similarity to known gentisate dioxygenases.
- Purified GDOxp formed a tetramer and exhibited dioxygenase activity against both gentisate and 1,4-dihydroxy-2-naphthoate, indicating broad substrate specificity.
- GDOxp lost activity and dissociated into monomers under unfavorable conditions but could be reactivated by ferrous ion addition.
- The N-terminal region was identified as important for tetramerization and enzyme stability.
Conclusions:
- GDOxp possesses broad substrate specificity, acting on both gentisate and a naphthoate derivative.
- The enzyme's activity is dependent on its tetrameric structure and the presence of ferrous ions.
- The N-terminus plays a key role in maintaining the functional tetrameric state of GDOxp.
- This study provides insights into the structure-function relationship of dioxygenases involved in aromatic compound degradation.
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