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Cryptic chlorination by a non-haem iron enzyme during cyclopropyl amino acid biosynthesis


Enzymatic incorporation of chlorine, bromine or iodine atoms occurs during the biosynthesis of more than 4,000 natural products1. Halogenation can have significant consequences for the bioactivity of these products so there is great interest in understanding the biological catalysts that perform these reactions. Enzymes that halogenate unactivated aliphatic groups have not previously been characterized. Here we report the activity of five proteins—CmaA, CmaB, CmaC, CmaD and CmaE—in the construction of coronamic acid (CMA; 1-amino-1-carboxy-2-ethylcyclopropane), a constituent of the phytotoxin coronatine synthesized by the phytopathogenic bacterium Pseudomonas syringae2. CMA derives from l-allo-isoleucine, which is covalently attached to CmaD through the actions of CmaA, a non-ribosomal peptide synthetase module, and CmaE, an unusual acyltransferase. We show that CmaB, a member of the non-haem Fe2+, α-ketoglutarate-dependent enzyme superfamily, is the first of its class to show halogenase activity, chlorinating the γ-position of l-allo-isoleucine. Another previously undescribed enzyme, CmaC, catalyses the formation of the cyclopropyl ring from the γ-Cl-l-allo-isoleucine product of the CmaB reaction. Together, CmaB and CmaC execute γ-halogenation followed by intramolecular γ-elimination, in which biological chlorination is a cryptic strategy for cyclopropyl ring formation.

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Figure 1: Biosynthesis of CMA and coronatine.
Figure 2: The transfer of l -Val from CmaA to CmaD catalysed by CmaE.
Figure 3: Analysis of the reactions catalysed by CmaB and CmaC.
Figure 4: Proposed mechanisms of CmaB and CmaC.

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We thank M. R. Rondon for providing Pseudomonas syringae pv. tomato DC3000, and M. G. Thomas for discussion. This work was supported in part by an NIH grant (C.T.W.), a Merck-sponsored Fellowship of the Helen Hay Whitney Foundation (F.H.V.), an NSERC Postdoctoral Fellowship (F.H.V.), an NIH Medical Scientist Training Program Fellowship (E.Y.), a Jane Coffin Childs Memorial Fund for Medical Research Fellowship (D.A.V.), and an Irving S. Sigal Postdoctoral Fellowship (S.E.O.).

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Correspondence to Christopher T. Walsh.

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Supplementary information

Supplementary Methods

This file describes the detailed synthesis protocol of γ-Cl-L-valine and γ-Cl-L-allo-isoleucine, the cloning of the cma genes, the overproduction and purification of the Cma proteins, and the biochemical assays that were performed. (PDF 168 kb)

Supplementary Table S1

dThis table contains the sequences of the oligonucleotides used to amplify the cmaA/B/C/D/E genes from Pseudomonas syringae pv. tomato DC3000 genomic DNA. (PDF 80 kb)

Supplementary Table S2

This table contains the kinetic parameters obtained using the ATP-[32P]PPi exchange assay with highly pure CmaA. (PDF 79 kb)

Supplementary Figure S1

This figure describes the chemical steps performed in the syntheses of γ-Cl-L-valine and γ-Cl-L-allo-isoleucine. (PDF 195 kb)

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Vaillancourt, F., Yeh, E., Vosburg, D. et al. Cryptic chlorination by a non-haem iron enzyme during cyclopropyl amino acid biosynthesis. Nature 436, 1191–1194 (2005).

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