The thioesterase domain from a nonribosomal peptide synthetase as a cyclization catalyst for integrin binding peptides

Abstract

Nonribosomal peptide synthetases responsible for the production of macrocyclic compounds often use their C-terminal thioesterase (TE) domain for enzymatic cyclization of a linear precursor. The excised TE domain from the nonribosomal peptide synthetase responsible for the production of the cyclic decapeptide tyrocidine A, TycC TE, retains autonomous ability to catalyze head-to-tail macrocyclization of a linear peptide thioester with the native sequence of tyrocidine A and can additionally cyclize peptide analogs that incorporate limited alterations in the peptide sequence. Here we show that TycC TE can catalyze macrocyclization of peptide substrates that are dramatically different from the native tyrocidine linear precursor. Several peptide thioesters that retain a limited number of elements of the native peptide sequence are shown to be substrates for TycC TE. These peptides were designed to integrate an Arg-Gly-Asp sequence that confers potential activity in the inhibition of ligand binding by integrin receptors. Although enzymatic hydrolysis of the peptide thioester substrates is preferred over cyclization, TycC TE can be used on a preparative scale to generate both linear and cyclic peptide products for functional characterization. The products are shown to be inhibitors of ligand binding by integrin receptors, with cyclization and N ␣ -methylation being important contributors to the nanomolar potency of the best inhibitors of fibrinogen binding to ␣IIb␤3 integrin. This study provides evidence for TycC TE as a versatile macrocyclization catalyst and raises the prospect of using TE catalysis for the generation of diverse macrocyclic peptide libraries that can be probed for novel biological function.M acrocyclic structure is a desirable feature for biologically or pharmaceutically active compounds. Macrocyclization stabilizes products from degradation and decreases the conformational flexibility of a molecule compared with its linear analog by constraining it to a biologically active conformation (1). In the biosynthesis of macrocyclic natural products, such as the macrolactam antibiotic tyrocidine, the macrolactone antifungal fengycin, and the macrolactone siderophore enterobactin by nonribosomal peptide synthetases (NRPSs), a C-terminal thioesterase (TE) domain introduces this constraint by catalyzing cyclization of a linear precursor (2, 3). Analogous cyclases in polyketide synthetase assemblies generate erythromycin and ketolide antibiotics as well as other bioactive macrolactones (4, 5).We have recently shown that several 28-to 35-kDa C-terminal TE domains excised from megadalton NRPS subunit proteins retain autonomous macrocyclization activity with peptide thioester substrates that mimic their natural protein-bound peptide phosphopantetheinyl substrates (6, 7). As a representative example, TycC TE, the C-terminal TE domain from the synthetase responsible for the biosynthesis of tyrocidine, macrocyclizes a soluble decapeptide thioester with the native peptide sequence of the antibiotic tyrocidine A (Fig. ...

Substrate recognition by nonribosomal peptide synthetase multi-enzymes

“…In these cases, however, no clear picture emerges from the results obtained, which are quite different for the two Te domains examined. Overall, Srf-Te appears to be relatively specific for its natural substrate, surfactin (Tseng et al, 2002), whereas Tyc-Te has a rather broad substrate specificity (Kohli et al, 2002b), which has allowed its use, in combination with solid phase peptide synthesis, for the creation of libraries of cyclic molecules, which can then be screened for biological activities (Kohli et al, 2002a).…”

Chemoenzymatic and Template‐Directed Synthesis of Bioactive Macrocyclic Peptides

“…Macrolactonization of Dap occurred with a k cat /K M only 1.3-fold reduced from that of the linear CDA analogue [113] and the ratio of cyclization to hydrolysis moderately dropped from 10:1 to 3:1 (Figure 6.5). This cyclization to hydrolysis ratio of Dap is still remarkable in comparison to studies with TycC TE, where simultaneous side chain alterations led to the predominance of hydrolysis over cyclization [75]. The efficient conversion to cyclic product may be caused by the Asp-D-Ala-Asp-Gly motif of the linear peptide precursor.…”

“…Walsh and co-workers showed that Tyc TE was capable to cyclize peptide substrates, in which up to 7 of 10 cognate residues were simultaneously replaced [75]. Macrolactamization of these linear peptide precursors containing an integrated RGD sequence yielded potent [76], which could be used to further optimize macrocyclic peptide/polyketide natural products, such as the immunosuppressant rapamycin and the anticancer agent epothilone [77].…”

“…Exchanges of C-and N-terminal residues of the linear peptide substrate often abolish formation of the macrocyclic product [5]. In other cases the yield of enzymatically generated ring formation dramatically suffers from linear hydrolytic byproducts due to nucleophilic attack of water molecules onto the acyl-enzyme intermediate [75]. Since the utility of TE domains strongly depends on substrate tolerance, efficient turnover, and high product yield, there is a constant need for engineered TE domains.…”

Chemoenzymatic and Template-Directed Synthesis of Bioactive Macrocyclic Peptides

“…Kohli and coworkers showed that Tyc TE was capable to cyclize peptide substrates, in which up to 7 of 10 cognate residues were simultaneously replaced (66). Macrolactamization of these linear peptide precursors containing an integrated RGD sequence yielded potent inhibitors of ligand binding by integrin receptors, with cyclization and N-methylation being important contributors to nanomolar potency (Fig.…”