Large scale purification of 1,2 Dehydroreticuline reductase from the opium poppy seedlings

Abstract

1,2-Dehydroreticuline reductase is an enzyme present in Papaver somniferum L. seedlings. It catalyses the hydrogenation of 1,2-dehydroreticuline by using NADPH to form (R)-reticuline, a key intermediate in the morphine biosynthetic pathway. The radioactive labelled [N-C3H3]- 1,2-dehydroreticuline was synthesized enzymatically from (S)-norreticuline by using two consecutive reactions catalysed by Berberis stolonifera enzymes, namely (S)- tetrahydroprotoberberine oxidase and N-methyltransferase, respectively. The [N-C3H3]-1,2- dehydroreticuline was used as substrate for assaying the enzyme activity during a large scale purification of 1,2-dehydroreticuline reductase from P. somniferum seedlings. The complete purification procedure included 6 steps: 55-85% ammonium sulfate fractionation, Phenyl Sepharose CL-4B, DEAE Sephacel, the first MonoQ, Superosel2 and the second MonoQ. The final enzyme preparation gave a single band on SDS-PAGE and was purified with 1433-fold and a 0.002% yield. The molecular weight of the reductase enzyme based on SDS-PAGE and gel filtration on Superose 12 was 34 kD. In this study, we also reported the partial amino acid sequence of 1,2-dehydroreticuline reductase.