Alkaloid binding to opium poppy major latex proteins triggers structural modification and functional aggregation.

Opium poppy accumulates copious amounts of several benzylisoquinoline alkaloids including morphine, noscapine, and papaverine, in the specialized cytoplasm of laticifers, which compose an internal secretory system associated with phloem throughout the plant. The contiguous latex includes an abundance of related proteins belonging to the pathogenesis-related (PR)10 family known collectively as major latex proteins (MLPs) and representing at least 35% of the total cellular protein content. Two latex MLP/PR10 proteins, thebaine synthase and neopione isomerase, have recently been shown to catalyze late steps in morphine biosynthesis previously assigned as spontaneous reactions. Using a combination of sucrose density-gradient fractionation-coupled proteomics, differential scanning fluorimetry, isothermal titration calorimetry, and X-ray crystallography, we show that the major latex proteins are a family of alkaloid-binding proteins that display altered conformation in the presence of certain ligands. Addition of MLP/PR10 proteins to yeast strains engineered with morphine biosynthetic genes from the plant significantly enhanced the conversion of salutaridine to morphinan alkaloids.

Structural studies of codeinone reductase reveal novel insights into aldo-keto reductase function in benzylisoquinoline alkaloid biosynthesis.

Benzylisoquinoline alkaloids (BIAs) are a class of specialized metabolites with a diverse range of chemical structures and physiological effects. Codeine and morphine are two closely related BIAs with particularly useful analgesic properties. The aldo-keto reductase (AKR) codeinone reductase (COR) catalyzes the final and penultimate steps in the biosynthesis of codeine and morphine, respectively, in opium poppy (Papaver somniferum). However, the structural determinants that mediate substrate recognition and catalysis are not well defined. Here, we describe the crystal structure of apo-COR determined to a resolution of 2.4 Å by molecular replacement using chalcone reductase as a search model. Structural comparisons of COR to closely related plant AKRs and more distantly related homologues reveal a novel conformation in the ß1α1 loop adjacent to the BIA-binding pocket. The proximity of this loop to several highly conserved active-site residues and the expected location of the nicotinamide ring of the NADP(H) cofactor suggest a model for BIA recognition that implies roles for several key residues. Using site-directed mutagenesis, we show that substitutions at Met-28 and His-120 of COR lead to changes in AKR activity for the major and minor substrates codeinone and neopinone, respectively. Our findings provide a framework for understanding the molecular basis of substrate recognition in COR and the closely related 1,2-dehydroreticuline reductase responsible for the second half of a stereochemical inversion that initiates the morphine biosynthesis pathway.