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Heterologous expression of alkene monooxygenase from Rhodococcus rhodochrous B-276
T J Smith1, J S Lloyd, S C Gallagher
1Department of Biological Sciences, University of Warwick, Coventry, UK.
European Journal of Biochemistry
|March 30, 1999
Summary
Alkene monooxygenase (AMO) enzyme activity was successfully expressed in Streptomyces lividans. This breakthrough enables detailed structure-function studies of AMO, crucial for understanding alkene epoxidation.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Alkene monooxygenase (AMO) from Rhodococcus rhodochrous B-276 is a multi-component enzyme system.
- AMO catalyzes stereoselective epoxidation of aliphatic alkenes, producing R enantiomers.
- Its active site is a dinuclear iron center, similar to methane monooxygenases.
Purpose of the Study:
- To achieve functional expression of the AMO complex in a heterologous host.
- To enable structure-function studies of AMO through site-directed mutagenesis.
Main Methods:
- Cloning the amoABCD operon into the pIJ6021 expression plasmid for Streptomyces lividans.
- Assessing AMO activity in cell-free extracts of S. lividans.
- Expressing individual AMO components (AmoB, AmoD) in Escherichia coli as glutathione S-transferase fusions.
Main Results:
- Functional AMO expression was achieved in S. lividans, with activity comparable to the wild-type organism.
- Recombinant AMO produced R-epoxypropane with high enantiomeric excess, stimulated by NADH.
- Individual functional coupling protein (AmoB) and reductase (AmoD) were successfully expressed in E. coli.
Conclusions:
- Streptomyces lividans is a suitable host for expressing the functional alkene monooxygenase complex.
- The developed expression systems facilitate future structure-function investigations of AMO via site-directed mutagenesis.