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The Pseudomonas oleovorans alkane hydroxylase gene. Sequence and expression
M Kok1, R Oldenhuis, M P van der Linden
1Groningen Biotechnology Center, University of Groningen, The Netherlands.
The Journal of Biological Chemistry
|April 5, 1989
Summary
Researchers identified and sequenced the Pseudomonas alkane hydroxylase gene (alkB). This gene encodes a membrane protein expressed in E. coli and Pseudomonas, crucial for alkane metabolism.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- The Pseudomonas OCT plasmid harbors genes for alkane metabolism.
- Understanding the alkane hydroxylase gene (alkB) is key to elucidating this metabolic pathway.
Purpose of the Study:
- To identify and sequence the Pseudomonas OCT plasmid-encoded alkane hydroxylase gene (alkB) and its promoter.
- To characterize the expression and protein structure of alkane hydroxylase.
Main Methods:
- Gene sequencing and promoter analysis.
- Nuclease S1 mapping to determine transcription initiation sites.
- Protein purification and amino-terminal sequencing.
- Homology analysis with other Pseudomonas promoters.
Main Results:
- The alkB gene and its promoter were identified and sequenced.
- The transcription initiation site of alkBAC mRNA was determined.
- The alkB gene product is a 401-amino acid polypeptide expressed at high levels in E. coli and Pseudomonas.
- The protein contains nine hydrophobic regions, with eight predicted to be membrane-spanning segments.
Conclusions:
- The alkB gene encodes a functional alkane hydroxylase integral membrane protein.
- The protein's structure is consistent with its role in alkane metabolism.
- High-level expression in heterologous hosts is feasible for further study.