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

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 compounds(1,2). Type I thioesterases (TEI) catalyse the release step from the assembly line(3) 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) domains(4-9). 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 systems(10-12). 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 bacteria(13). 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 enzymes(3,14) 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.