The chloroalkaloid (−)-acutumine is biosynthesized via a Fe(II)- and 2-oxoglutarate-dependent halogenase in Menispermaceae plants

Abstract

Plant halogenated natural products are rare and harbor various interesting bioactivities, yet the biochemical basis for the involved halogenation chemistry is unknown. While a handful of Fe(II)-and 2-oxoglutarate-dependent halogenases (2ODHs) have been found to catalyze regioselective halogenation of unactivated C-H bonds in bacteria, they remain uncharacterized in the plant kingdom. Here, we report the discovery of dechloroacutumine halogenase (DAH) from Menispermaceae plants known to produce the tetracyclic chloroalkaloid (−)-acutumine. DAH is a 2ODH of plant origin and catalyzes the terminal chlorination step in the biosynthesis of (−)-acutumine. Phylogenetic analyses reveal that DAH evolved independently in Menispermaceae plants and in bacteria, illustrating an exemplary case of parallel evolution in specialized metabolism across domains of life. We show that at the presence of azide anion, DAH also exhibits promiscuous azidation activity against dechloroacutumine. This study opens avenues for expanding plant chemodiversity through halogenation and azidation biochemistry.

Halogenases: a palette of emerging opportunities for synthetic biology–synthetic chemistry and C–H functionalisation

“…(A) Known substrate scope of the NHFe halogense BesD and enzymes from the BesD family, 84 (B) AdeV chlorination of 2 0 -dAMP in adechlorin biosynthesis and other known substrates of AdeV, 85 (C) SaDAH chlorination in acutumine biosynthesis. 181 the chlorinated natural product adechlorin 260 in Actinomadura sp. ATCC 39365, opening up the path to access valuable halonucleosides 85 (Scheme 10B).…”

“…Fatty acid derivatives of fluoroacetate such as o-fluorooleic acid accumulating in the seeds of Dichapetalum toxicarium. 181 An exciting new NHFe halogenase DAH that performs the stereoselective late stage chlorination of the complex alkaloid dechloroacutimine 265, (Scheme 10C) produced by menispermaceae plants, has been found and its activity demonstrated in vitro. This represents the very first NHFeHal found in plants.…”

Halogenases for biosynthetic pathway engineering: Toward new routes to naturals and non-naturals

“…[175] Dechloroacutumine halogenase (DAH) catalyzes the terminal chlorination step in the biosynthesis of acutumine by chlorinating dechloroacutumine 79 to acutumine 77. [176] Both the enzymes have a low sequence similarity with other α-KG-Hals which has potential utility in expanding chemo diversity of nucleotides and acutumine type scaffolds. There are some excellent reviews on the enzymology and halogenation mechanism of α-KG-Hal and α-KGO superfamily of enzymes for further reading.…”

Nature's Chemists: The Discovery and Engineering of Phytochemical Biosynthesis

“…Protein expression in E. coli is often followed by enzyme purification and in vitro assays. This enables the assessment of the biochemical activities of the candidate enzymes outside the complex cellular context (Caputi et al, 2018 ; Torrens-Spence et al, 2018 ; Lichman et al, 2019 ; Kim et al, 2020 ) ( Figure 3 ).…”