Structural Basis for the Cyclization of the Lipopeptide Antibiotic Surfactin by the Thioesterase Domain SrfTE

Abstract

Many biologically active natural peptides are synthesized by nonribosomal peptide synthetases (NRPS). Product release is accomplished by dedicated thioesterase (TE) domains, some of which catalyze an intramolecular cyclization to form macrolactone or macrolactam cyclic peptides. The excised 28 kDa SrfTE domain, a member of the alpha/beta hydrolase enzyme family, exhibits a distinctive bowl-shaped hydrophobic cavity that hosts the acylpeptide substrate and tolerates its folding to form a cyclic structure. A substrate analog confirms the substrate binding site and suggests a mechanism for substrate acylation/deacylation. Docking of the peptidyl carrier protein domain immediately preceding SrfTE positions the 4'-phosphopantheinyl prosthetic group that transfers the nascent acyl-peptide chain to SrfTE. The structure provides a basis for understanding the mechanism of acyl-PCP substrate recognition and for the cyclization reaction that results in release of the macrolactone cyclic heptapeptide.

Structure and function of an iterative polyketide synthase thioesterase domain catalyzing Claisen cyclization in aflatoxin biosynthesis

“…TEs bear the canonical TE catalytic triad consisting of active site Ser-His-Asp (15) residues, which were tentatively identified in the PksA TE/CLC domain by primary sequence comparisons and confirmed here in mutagenesis studies and the crystal structure demonstrating their involvement in Claisen cyclization. The structure shows that fungal CLCs belong to the α/β-hydrolase family but harbor a deep, hydrophobic substrate-binding chamber that is distinct from previously reported TE structures from bacterial modular PKSs (16)(17)(18), nonribosomal peptide synthetases (NRPSs) (19,20) and human FAS (21,22). The structure-function studies presented herein provide a picture that is likely general for C-C bond formation in polyketide chain-terminating CLCs.…”

“…Such a lid is also seen in the DEBS (17), PICS (18), surfactin synthetase (SrfA-C) (19), fengycin synthetase (FenB) (20), and enterobactin (EntF) (24) TE structures, although there is considerable variability among these individual lid regions. A second small region of structural variability between these enzymes and PksA TE is found in the loop between β7 and the vestigial αE.…”

Structure and Function of the RedJ Protein, a Thioesterase from the Prodiginine Biosynthetic Pathway in Streptomyces coelicolor

“…Crystal and NMR structures of TE IIs and NRPS TE Is demonstrate that the helical lid domain in these monomeric TEs is mobile and may control access of substrates to the active site (13,(21)(22)(23)25). The crystal structures of RedJ also demonstrate lid mobility.…”

“…Active Site Entrance Channel-A flexible entrance channel for phosphopantetheine-linked substrates has been identified between the core and lid of other monomeric TEs (13,(21)(22)(23)25). The analogous region of RedJ also forms a channel into the active site (Fig.…”

“…However, for TE IIs and NRPS TE Is, the lid region is flexible based on crystal and NMR solution structures that captured distinct conformations and movement of the lid (13,(21)(22)(23)25). Lid movement has been proposed to regulate access of substrates to the active site, to recognize specific ACPs, and to change the size and shape of the substrate chamber (13,(21)(22)(23)25). Furthermore, TE quaternary structures vary.…”