Molecular cloning and functional expression of codeinone reductase: the penultimate enzyme in morphine biosynthesis in the opium poppy Papaver somniferum

The narcotic analgesic morphine is the major alkaloid of the opium poppy Pa paver somniferum. Its biosynthetic precursor codeine is currently the most widely used and effective antitussive agent. Along the morphine biosyntheti c pathway in opium poppy, codeinone reductase catalyzes the NADPH-dependent reduction of codeinone to codeine. In this study, we have isolated and cha racterized four cDNAs encoding codeinone reductase isoforms and have functi onally expressed them in Escherichia coli. Heterologously expressed codeino ne reductase-calmodulin-binding peptide fusion protein was purified from E. coli using calmodulin affinity column chromatography in a yield of 10 mg e nzyme I-1. These four isoforms demonstrated very similar physical propertie s and substrate specificity. As least six alleles appear to be present in t he poppy genome. A comparison of the translations of the nucleotide sequenc es indicate that the codeinone reductase isoforms are 53% identical to 6'-d eoxychalcone synthase from soybean suggesting an evolutionary although not a functional link between enzymes of phenylpropanoid and alkaloid biosynthe sis. By sequence comparison, both codeinone reductase and 6'-deoxychalcone synthase belong to the aldo/keto reductase family, a group of structurally and functionally related NADPH-dependent oxidoreductases, and thereby possi bly arise from primary metabolism.