Structural basis for the selectivity of the external thioesterase of the surfactin synthetase

Abstract

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) are found in bacteria, fungi, and plants, in the main, producing antibiotics. They are macromolecular machines that rely on the activity of thioesterases to produce biologically active small molecules. They are of particular interest as an assembly system that might be adapted for the production of novel bioactive compounds with possible therapeutic activity. Frueh et al. have solved the structure of a carrier protein — part of the EntF NRPS subunit of enterobactin synthetase — bound to a type I thioesterase. (Type I thioesterases catalyse the final 'release' step of the small molecule from the NRPS or PKS machinery.) The structure reveals that part of the thioesterase can flip open to reveal the carrier-protein binding site of the enzyme; this movement allows the tether of the carrier protein to access the active site. Koglin et al. determined the structure of conformational sub-states of a thioesterase II enzyme. Type II thioesterases are required to regenerate a functional 4'-phosphopantetheine cofactor when it gets mis-primed by reacting with acetyl- and short chain acyl-residues. Comparison with the structures of type I thioesterases reveals the basis for substrate selectivity and the different modes of interaction of the two types of thioesterases with thiolation domains. The three-dimensional structure of a type II thioesterase, free and in complex with a thiolation domain, is determined. Comparison with type I thioesterases reveal the basis for substrate selectivity and the different modes of interaction of the two types of thioesterases with thiolation domains. Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) found in bacteria, fungi and plants use two different types of thioesterases for the production of highly active biological compounds1,2. Type I thioesterases (TEI) catalyse the release step from the assembly line3 of the final product where it is transported from one reaction centre to the next as a thioester linked to a 4′-phosphopantetheine (4′-PP) cofactor that is covalently attached to thiolation (T) domains4,5,6,7,8,9. The second enzyme involved in the synthesis of these secondary metabolites, the type II thioesterase (TEII), is a crucial repair enzyme for the regeneration of functional 4′-PP cofactors of holo-T domains of NRPS and PKS systems10,11,12. Mispriming of 4′-PP cofactors by acetyl- and short-chain acyl-residues interrupts the biosynthetic system. This repair reaction is very important, because roughly 80% of CoA, the precursor of the 4′-PP cofactor, is acetylated in bacteria13. Here we report the three-dimensional structure of a type II thioesterase from Bacillus subtilis free and in complex with a T domain. Comparison with structures of TEI enzymes3,14 shows the basis for substrate selectivity and the different modes of interaction of TEII and TEI enzymes with T domains. Furthermore, we show that the TEII enzyme exists in several conformations of which only one is selected on interaction with its native substrate, a modified holo-T domain.