No abstract

Enzymatic incorporation of chlorine, bromine or iodine atoms occurs during the biosynthesis of more than 4,000 natural products. 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 syringae. 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 Fe(2+), alpha-ketoglutarate-dependent enzyme superfamily, is the first of its class to show halogenase activity, chlorinating the gamma-position of l-allo-isoleucine. Another previously undescribed enzyme, CmaC, catalyses the formation of the cyclopropyl ring from the gamma-Cl-l-allo-isoleucine product of the CmaB reaction. Together, CmaB and CmaC execute gamma-halogenation followed by intramolecular gamma-elimination, in which biological chlorination is a cryptic strategy for cyclopropyl ring formation.

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties 1 . Like fatty acid synthases (FAS), these enzymes are organized in modular assembly lines in which each module, made of conserved domains, incorporates a given monomer unit into the growing chain. Knowledge about domain or module interactions may enable reengineering of this assembly line enzymatic organization and open avenues for the design of new bioactive compounds with improved therapeutic properties. So far, little structural information has been available on how the domains interact and communicate. This may be because of inherent interdomain mobility hindering crystallization, or because crystallized molecules may not represent the active domain orientations 2 . In solution, the large size and internal dynamics of multidomain fragments (>35 kilodaltons) make structure determination by nuclear magnetic resonance a challenge and require advanced technologies. Here we present the solution structure of the apo-thiolation-thioesterase (T-TE) di-domain fragment of the Escherichia coli enterobactin synthetase EntF NRPS subunit. In the holoenzyme, the T domain carries the growing chain tethered to a 4′-phosphopantetheine whereas the TE domain catalyses hydrolysis and cyclization of the iron chelator enterobactin. The T-TE di-domain forms a compact but dynamic structure with a well-defined domain interface; the two active sites are at a suitable distance for substrate transfer from T to TE. We observe extensive interdomain and intradomain motions for well-defined regions and show that these are modulated by interactions with proteins that participate in the biosynthesis. The T-TE interaction described here provides a model for NRPS, PKS and FAS function in general as T-TE-like di-domains typically catalyse the last step in numerous assembly-line chain-termination machineries.The Escherichia coli enterobactin synthetase (Ent) synthesizes the siderophore enterobactin, a virulence factor used by E. coli to infect iron-limited microenvironments of vertebrate hosts. Enterobactin is obtained by three iterative condensations of dihydroxybenzoate (DHB) with serine, followed by macrocyclizing release of the macrolactone that can form high-affinity hexadentate complexes with ferric iron 3,4 (Fig. 1). Ent is a two-module (EntB/EntE and EntF), three-protein assembly line that combines the features of both a type I NRPS, in which all domains interact in cis in a single protein chain (EntF), and a type II NRPS, in which the (Fig. 1a). The EntE and EntF adenylation (A) domains select the appropriate substrates (DHB or serine) and activate them as acyl-adenosinemonophosphates. These are then loaded on the 4′-PP arms attached to Ser 245 of the holo-EntB aryl carrier protein (ArCP) and to Ser 48 of holo-EntF T domains, respectively (Fig. 1b). The EntF condensation (C) domain catalyses the DHB-Ser amide-bond formation (Fig. 1c). The DHB-Ser chains are next transferre...