Tropinone synthesis via an atypical polyketide synthase and P450-mediated cyclization

Matthew A Bedewitz,A Daniel Jones,John C D’Auria,Cornelius S Barry

doi:10.1038/s41467-018-07671-3

Matthew A Bedewitz, A Daniel Jones + Show 2 more

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https://doi.org/10.1038/s41467-018-07671-3

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Abstract

Tropinone is the first intermediate in the biosynthesis of the pharmacologically important tropane alkaloids that possesses the 8-azabicyclo[3.2.1]octane core bicyclic structure that defines this alkaloid class. Chemical synthesis of tropinone was achieved in 1901 but the mechanism of tropinone biosynthesis has remained elusive. In this study, we identify a root-expressed type III polyketide synthase from Atropa belladonna (AbPYKS) that catalyzes the formation of 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid. This catalysis proceeds through a non-canonical mechanism that directly utilizes an unconjugated N-methyl-Δ1-pyrrolinium cation as the starter substrate for two rounds of malonyl-Coenzyme A mediated decarboxylative condensation. Subsequent formation of tropinone from 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid is achieved through cytochrome P450-mediated catalysis by AbCYP82M3. Silencing of AbPYKS and AbCYP82M3 reduces tropane levels in A. belladonna. This study reveals the mechanism of tropinone biosynthesis, explains the in planta co-occurrence of pyrrolidines and tropanes, and demonstrates the feasibility of tropane engineering in a non-tropane producing plant.

Highlights

Tropinone is the first intermediate in the biosynthesis of the pharmacologically important tropane alkaloids that possesses the 8-azabicyclo[3.2.1]octane core bicyclic structure that defines this alkaloid class
The identification of 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid as an intermediate in tropinone formation led to the hypothesis that a polyketide synthase is involved in the synthesis of tropinone from the pyrrolinium cation following two rounds of decarboxylative condensation by malonyl-CoA5
4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid, which is the predicted metabolite obtained from the N-methyl-Δ1-pyrrolinium cation following two rounds of PKS-mediated elongation using malonylCoA, is reduced by 92% in TRV2:ab1322 lines

Summary

Introduction

Tropinone is the first intermediate in the biosynthesis of the pharmacologically important tropane alkaloids that possesses the 8-azabicyclo[3.2.1]octane core bicyclic structure that defines this alkaloid class. In vivo labeling with radiolabeled substrates identified ornithine as a precursor of tropanes in the Solanaceae[13], with similar experimental approaches utilized to delineate the pathway intermediates from ornithine to tropinone, and in particular to identify 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid (5) as an intermediate pathway metabolite between the N-methyl-Δ1-pyrrolinium cation (4) and tropinone[5,14,15,16] The existence of this pathway is largely accepted and the enzymes that catalyze the steps from ornithine to the N-methyl-Δ1-pyrrolinium cation, the first ring closure, are known and the corresponding genes are preferentially expressed in roots, the site of tropane alkaloid biosynthesis[17] (Fig. 1a). These data reveal the biological route to formation of tropinone, a metabolite first synthesized chemically over a century ago

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