Detection of substrate binding motifs for morphine biosynthetic pathway intermediates in novel wound inducible (R,S)-reticuline 7-O-methyltransferase of Papaver somniferum

Abstract

The benzylisoquinoline alkaloids (BIA) comprise a large and diverse group of nitrogen-containing secondary metabolites with about 2500 compounds identified in plants. BIA biosynthesis begins with the condensation of the tyrosine derived precursors dopamine and p-hydroxyphenylacetaldehyde to (S)-norcoclaurine. Subsequent regiospecific O- and N-methylations and aromatic ring hydroxylation lead to (S)-reticuline, which is the central intermediate for almost all BIAs. For morphinan alkaloid biosynthesis, (S)-reticuline undergoes an inversion of stereochemistry to (R)-reticuline, followed by C-C phenol coupling catalyzed by a unique cytochrome P450-dependent monooxygenase to yield salutaridine. The cDNA sequence of enzymes leading to (S)-reticuline, as well as those involved in the conversion of (R)-reticuline to salutaridine-7-O-acetate are already characterized. The inversion of (S)-reticuline to (R)-reticuline represent the important steps in morphine biosynthesis. Wound induced transcript accumulation in Papaver reveals a novel wound inducible EST (NCBI DbEST: GO238757) showing homology with (R,S)-reticuline 7-O-methyltransferase (ID: Q6WUC2) isolated from Papaver somniferum. We compare the substrate binding homology of this novel wound inducible (R,S)-reticuline 7-O-methyltransferase (7-OMT) using template of P. somniferum (Q6WUC2; gb|AAQ01668) as experimental control. Homology modeling with 70% identity & 85% similarity with catalytic site of template protein i.e., (Q6WUC2) short chain dehydrogenase/reductase (SDR), showed docking energy -69.9 and -75.8 kcal/mol with (S)-Reticuline (CID:439653) and (R)-Reticuline (CID:440586) respectively, which are comparable with experimental control binding site interaction energies. Docking of S- & R-reticuline into the active site revealed eight (F(5), E(18), W(24), C(47), F(44), P(45), C(46) and I(47) amino acids presumably responsible for the high substrate specificity of (R,S)-reticuline 7-O-methyltransferase