Regeneration of misprimed nonribosomal peptide synthetases by type II thioesterases

Abstract

Nonribosomal peptide synthetases (NRPSs) assemble structurally complex peptides from simple building blocks such as amino and carboxyl acids. Product release by macrocyclization or hydrolysis is catalyzed by a thioesterase domain that is an integrated part of the NRPS enzyme. A second thioesterase of type II (TEII) encoded by a distinct gene associated with the NRPS cluster was previously shown by means of gene disruption to be important for efficient product formation. However, the actual role of TEIIs in nonribosomal peptide synthesis remained obscure. Here we report the biochemical characterization of two such TEII enzymes that are associated with the synthetases of the peptide antibiotics surfactin (TEII(srf)) and bacitracin (TEII(bac)). Both enzymes were shown to efficiently regenerate misacylated thiol groups of 4'-phosphopantetheine (4'PP) cofactors attached to the peptidyl carrier proteins (PCPs) of NRPSs. For TEII(srf), a K(M) of 0.9 microM and a k(cat) of 95 min(-1) was determined for acetyl-PCP hydrolysis. Both enzymes could also hydrolyze aminoacyl or peptidyl PCPs, intermediates of nonribosomal peptide synthesis. However, this reaction is unlikely to be of physiological relevance. Similar intermediates of the primary metabolism such as CoA derivatives and acetyl-acyl carrier proteins of fatty acid synthesis were also not significantly hydrolyzed, as investigated with TEII(srf). These findings support a model in which the physiological role of TEIIs in nonribosomal peptide synthesis is the regeneration of misacylated NRPS, which result from the apo to holo conversion of NRPS enzymes because of the promiscuity of dedicated 4'PP transferases that use not only free CoA, but also acyl-CoAs as 4'PP donors.

Fig 1.

(a) Structures of the peptide antibiotics surfactin, tyrocidine, and bacitracin. (b) Organization of the corresponding gene clusters. Genes encoding the TEIIs are highlighted in gray.

Fig 2.

Overview of thioesterase assays. (a) Proline quench assay. (b) Acetyl-S-4′PP-NRPS hydrolysis assay. (c) Acetyl-S-4′PP-ACP hydrolysis assay. (Left) A schematic drawing of the corresponding assays. Compounds shown in red carry a radioactive label. In all cases, a NRPS/thioesterase ratio of 1:0.5 was used.

Fig 3.

HPLC analysis of the TEII_srf-catalyzed acetyl-4′PP-PCP hydrolysis. The identities of the compounds acetyl-S-4′PP-PCP, TNB-S-4′PP-PCP, and apo-PCP were confirmed by MS.

Fig 4.

In vitro simulation of the deblocking after mispriming model by a tripeptide formation assay using correctly primed, acetyl-CoA misprimed, and acetyl-CoA misprimed NRPSs in the presence of TEII_srf.

Fig 5.

The deblocking after mispriming model for the role of TEIIs in nonribosomal peptide assembly. Apo to holo conversion of NRPSs is catalyzed by dedicated 4′PP transferases that accept CoA and acyl-CoA as substrates. If acyl-4′PP is the transferred cofactor, a TEII then hydrolyzes the acyl group and thereby regenerates a misprimed NRPS to the active free holo form.