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Nucleotide sequence and over-expression of morphine dehydrogenase, a plasmid-encoded gene from Pseudomonas putida M10
D L Willey1, D A Caswell, C R Lowe
1Institute of Biotechnology, University of Cambridge, U.K.
Abstract:
Pseudomonas putida M10 was originally isolated from factory waste liquors by selection for growth on morphine. The NADP(+)-dependent morphine dehydrogenase that initiates morphine catabolism is encoded by a large plasmid of 165 kb. Treatment of P. putida M10 with ethidium bromide led to the isolation of a putative plasmid-free strain that was incapable of growth on morphine. The structural gene for morphine dehydrogenase, morA, has been located on the plasmid by oligonucleotide hybridization, by coupled transcription-translation of cloned restriction fragments and by nucleotide sequence analysis and is contained within a 1.7 kb SphI fragment that has been cloned into Escherichia coli. The cloned dehydrogenase enzyme is expressed at high levels in E. coli resulting in a 65-fold increase in morphine dehydrogenase activity in cell-free extracts compared with P. putida M10. Morphine dehydrogenase was rapidly purified to homogeneity, as judged by SDS/PAGE, by a one-step affinity chromatography procedure on Mimetic Orange 3 A6XL. The properties of the purified enzyme were identical with those previously reported for P. putida M10 morphine dehydrogenase. The morA gene was sequenced and the deduced amino acid sequence confirmed by N-terminal amino acid sequencing of the over-expressed protein. The predicted amino acid sequence of morA, deduced from the nucleotide sequence, indicated that morphine dehydrogenase did not belong to the non-metal-requiring short-chain class of dehydrogenases, but was more closely related to the aldo-ketoreductases.
Insights
Pseudomonas putida M10 utilizes a plasmid-encoded enzyme, morphine dehydrogenase, for morphine breakdown. Researchers cloned the gene (morA) and overexpressed the enzyme in E. coli, confirming its properties and classification.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Pseudomonas putida M10 degrades morphine using a specific enzyme.
- This enzyme, morphine dehydrogenase, is encoded on a large plasmid.
- Loss of the plasmid renders the bacteria unable to metabolize morphine.
Purpose of the Study:
- To identify and characterize the gene encoding morphine dehydrogenase.
- To clone and express the morphine dehydrogenase gene in a heterologous host.
- To determine the enzymatic properties and classification of morphine dehydrogenase.
Main Methods:
- Isolation of a plasmid-free strain of P. putida M10.
- Oligonucleotide hybridization and coupled transcription-translation for gene localization.
- Cloning of the morA gene into Escherichia coli and subsequent enzyme expression.
- Purification of morphine dehydrogenase using affinity chromatography.
- Nucleotide and amino acid sequencing of the morA gene and protein.
Main Results:
- The morphine dehydrogenase gene (morA) was localized to a 1.7 kb SphI fragment on the plasmid.
- Cloning and expression in E. coli resulted in a 65-fold increase in enzyme activity.
- Purified morphine dehydrogenase exhibited properties consistent with the native enzyme.
- Sequence analysis indicated that morphine dehydrogenase is related to aldo-ketoreductases, not short-chain dehydrogenases.
Conclusions:
- The morA gene responsible for morphine dehydrogenase is located on the P. putida M10 plasmid.
- Heterologous expression in E. coli is an effective method for high-level production of active morphine dehydrogenase.
- Morphine dehydrogenase represents a novel member of the aldo-ketoreductase family.