Codeinone reductase isoforms with differential stability, efficiency and product selectivity in opium poppy. (21st June 2018)

• Record Type: Journal Article
• Authors: Dastmalchi, Mehran; Chang, Limei; Torres, Miguel A.; Ng, Kenneth K. S.; Facchini, Peter J.
• Is Part Of: Plant journal. Volume 95:Number 4(2018)
• Page Start: 631
• Page End: 647
• Publication Date: 2018-06-21
• Subjects: benzylisoquinoline alkaloid -- codeinone reductase -- enzyme isoforms -- codeine -- neopine -- morphine -- neomorphine -- opiate alkaloid biosynthesis -- Papaver somniferum; Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.13975
• Languages: English
• ISSNs: 0960-7412

Abstract

Summary: Codeinone reductase (COR) catalyzes the reversible NADPH‐dependent reduction of codeinone to codeine as the penultimate step of morphine biosynthesis in opium poppy ( Papaver somniferum ). It also irreversibly reduces neopinone, which forms by spontaneous isomerization in aqueous solution from codeinone, to neopine. In a parallel pathway involving 3‐ O ‐demethylated analogs, COR converts morphinone to morphine, and neomorphinone to neomorphine. Similar to neopine, the formation of neomorphine by COR is irreversible. Neopine is a minor substrate for codeine O ‐demethylase (CODM), yielding morphine. In the plant, neopine levels are low and neomorphine has not been detected. Silencing of CODM leads to accumulation of upstream metabolites, such as codeine and thebaine, but does not result in a shift towards higher relative concentrations of neopine, suggesting a mechanism in the plant for limiting neopine production. In yeast ( Saccharomyces cerevisiae ) engineered to produce opiate alkaloids, the catalytic properties of COR lead to accumulation of neopine and neomorphine as major products. An isoform (COR‐B) was isolated from opium poppy chemotype Bea's Choice that showed higher catalytic activity than previously characterized CORs, and it yielded mostly neopine in vitro and in engineered yeast. Five catalytically distinct COR isoforms (COR1.1–1.4 and COR‐B) were used to determine sequence–function relationships that influence product selectivity. Biochemical characterization and site‐directed mutagenesis of native COR isoforms identified four residues (V25, K41, F129 and W279) that affected protein stability, reaction velocity, and product selectivity and output. Improvement of COR performance coupled with an ability to guide pathway flux is necessary to facilitate commercial production of opiate alkaloids in engineered microorganisms. Significance Statement: Codeinone reductase (COR) catalyzes a pivotal conversion in the biosynthesis of morphine in opium poppy. We correct previous conclusions regarding the enzymology of COR and demonstrate the sensitivity of the enzyme to amino acid substitutions found in various isoforms. Our work helps to explain why the product selectivity coordinated by COR in the plant is not reflected in engineered yeast strains, which accumulate alternative compounds. Understanding COR is crucial for the reconstitution of opiate biosynthesis in microorganisms.