Nucleotide sequence and over-expression of morphine dehydrogenase, a plasmid-encoded gene from Pseudomonas putida M10

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.