Characterization of the Surfactin Synthetase C-Terminal Thioesterase Domain as a Cyclic Depsipeptide Synthase

Abstract

The C-terminal thioesterase domain of the nonribosomal peptide synthetase producing the lipopetide surfactin (Srf TE) retains autonomous ability to generate the cyclic peptidolactone skeleton of surfactin when provided with a soluble beta-hydroxy-butyryl-heptapeptidyl thioester substrate. Utilizing the recently solved crystal structure [Bruner, S. D., et al. (2002) Structure 10, 301-310], the active-site nucleophile, Ser80, was changed to Cys, and the other members of the catalytic triad, Asp107 and His207, were changed to Ala, with the resulting mutants lacking detectable activity. Two cationic side chains in the active site, Lys111 and Arg120, were changed to Ala, causing an increased partitioning of the product to hydrolysis, as did a P26G mutant, mimicking the behavior of lipases. To evaluate recognition elements in substrates used by Srf TE, alterations to the fatty acyl group, the heptapeptide, and the thioester leaving group were made, and the resulting substrates were characterized for kinetic competency and flux of product to cyclization or hydrolysis. Alterations that could be accepted for cyclization were identified in all three parts of the substrate, although tolerance limits for changes varied. In addition, cocrystal structures of Srf TE with dipeptidyl boronate inhibitors were solved, illustrating the critical binding determinants of the substrate. On the basis of the structures and biochemical data, the cyclizing conformation of the surfactin peptide was modeled into the enzyme active site.

Mutational analysis of a type II thioesterase associated with nonribosomal peptide synthesis

“…2, in the absence of TEII, a very slow ÔbackgroundÕ hydrolysis (decreasing enzyme-bound radioactivity) occurred over the time scale observed. However, the enzyme-bound radioactivity decreased rapidly on addition of S86C to an assay mixture containing [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]acetyl-S-Ppant-PCP (Fig. 2B), confirming the enzyme's functionality.…”

“…This inhibition of S86C is due to the covalent modification of the active-site Cys with TNB as judged by ESI-MS experiments. (B) Radioactive assay: [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]acetyl-4¢-S-Ppant-PCP is used as substrate for TEII srf as previously described [17]. Therefore, apo-PCP was converted into [1- C]acetyl-CoA as substrate.…”

“…The thiol group of this thioester belongs to 4¢-Ppant, the prosthetic group of the peptidyl carrier proteins (PCPs) and acyl carrier proteins. The post-translational modification (priming) of the carrier proteins is carried out by dedicated 4¢-phosphopantetheine transferases such as Sfp [6][7][8].Two types of thioesterase are associated with NRPSs and PKSs: the well-studied integrated type I thioesterase domains (TE domains), which are responsible for the release of the synthesized products from the enzymatic templates [9][10][11], and the external stand-alone type II thioesterases (TEIIs). Disruption of the corresponding TEII genes in the producer strains inhibited product formation by 80-90% [12][13][14].…”

Review of surfactin chemical properties and the potential biomedical applications

“…The intramolecular lactonization to the initiating β-OH fatty acyl moiety possibly involves a second thioesterase, i.e. the above mentioned SrfTE-II [46,47].…”

Absolute Configuration and Biosynthesis of Pahayokolide A from Lyngbya sp. Strain 15-2 of the Florida Everglades

“…In the structure of Srf TE (Fig. 2-8B), the three essential amino acids (Ser80, His207, and Asp107) for the enzymatic activity of the catalytic triad were identified by mutational analysis (Tseng, et al, 2002). With the exception of two positive charged side chains of Lys111 and Arg120, the cavity is bound mainly by hydrophobic residues.…”