Dynamic thiolation-thioesterase structure of a non-ribosomal peptide synthetase

Abstract

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties. 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. 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.

Figure 1. Summary of enterobactin synthesis

The grey, red and blue boxes represent individual domains; the C, A, T and TE domains are described in the text. The EntB ICL domain is an isochorismate ligase that participates in the conversion of chorismate to DHB, a preliminary step to the synthesis of enterobactin. Initially, EntD loads the phosphopantetheinyl arm (4′-PP, represented as a wavy line) onto the active-site serines of apo-EntB and apo-EntF. The synthesis steps (a–e) are described in the text.

Figure 2. Structure of the EntF T–TE fragment

a, A ribbon diagram is shown. The T domain (red) is wedged between the core (blue) and the lid (green) of the TE domain. Active sites are shown as yellow spheres. The double-headed arrow emphasizes that the flap formed by the α4_TE and α5_TE helices is relatively mobile, opening frequently, which seems to be necessary to accommodate the 4′-PP arm in the processes depicted in Fig. 1. b, Surface representation of the region containing the canyon (grey) which must open to accommodate the 4′-PP arm in the holoprotein. The cyclization bucket is shown in white and grey. In this conformation, Ser48Ala and Ser 180 are buried. c, The domain interface is shown. The side chains of all residues giving rise to interdomain nuclear Overhauser effects (NOEs) are shown.

Figure 3. Interaction with Sfp

a, Residues subject to two different environments. The colour code represents small (blue), medium (red) and large (yellow) chemical shift differences between the two forms as reported in b. c, Chemical shift differences between the major conformer of free T–TE and the excised TE domain. d–f, Spectral signatures of the Sfp interaction. d, The reference spectrum of Ser48Ala. The open form is only visible with longer acquisitions (Supplementary Fig. 3). On addition of Sfp, the signals of the residues in green (see a) disappear from the spectrum, and those of the second form appear (e). f, Corresponding region of the spectrum of the excised TE.

Figure 4. Interaction with the C domain

Many resonances experience shifts on addition of the C domain (bottom), indicating a modulation of the environment of the corresponding residues. Those belonging to the T domain form an interaction surface that does not overlap with the T–TE interface (top). Thus, no disruption of the T–TE interaction is observed. The effects on the TE domain may be due to weak, indirect interactions with the C domain, or to a secondary effect due to fluctuations of the dynamics or modifications of the conformations in these regions. Residues with shifts larger than one s.d. from the mean (red line, bottom) are colour coded with a gradation from white to red.