Chemoenzymatic and Template-Directed Synthesis of Bioactive Macrocyclic Peptides

Nonribosomal peptide synthetases (NRPS) are large multienzyme complexes, which simultaneously represent template and biosynthetic machinery for the production of structurally diverse peptidic products that feature high pharmacological and biological activities. A key determinant of nonribosomal pept...

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Bibliographic Details
Main Author: Grünewald, Jan
Contributors: Marahiel, Mohamed (Prof.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2005
Chemie
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Summary:Nonribosomal peptide synthetases (NRPS) are large multienzyme complexes, which simultaneously represent template and biosynthetic machinery for the production of structurally diverse peptidic products that feature high pharmacological and biological activities. A key determinant of nonribosomal peptide product activity is the common macrocyclic structure of many compounds. Macrocyclization is catalyzed in the last step of nonribosomal synthesis by thioesterase (TE) domain activity. The herein presented work describes the first biochemical characterization of a TE domain of a streptomycete, the thioesterase of the S. coelicolor calcium-dependent antibiotic (CDA) synthetase. This recombinant cyclase catalyzes macrolactone formation of linear peptidyl-thioesters based on a sequence analogous to natural CDA. For substrate mimics, the phosphopantetheine cofactor was successfully substituted by various thioester leaving groups. The best rates for cyclization were determined for the thiophenol leaving group, revealing that chemical reactivity is more important for enzyme acylation than cofactor recognition. Interestingly, CDA cyclase catalyzes the formation of two regioisomeric macrolactones, which arise from simultaneous nucleophilic attack of the two adjacent Thr2 and Ser1 residues onto the C-terminal Trp11 of the acyl-enzyme intermediate. To further explore this relaxed regioselectivity of CDA TE, alterations to the peptide backbone and the fatty acyl chain were made. Substitution of either Thr2 or Ser1 by alanine led to selective formation of a decapeptide or undecapeptide lactone ring. However, the stereoselectivity of CDA cyclase was fully retained, thus accepting only L-configured Ser1 and Thr2 for cyclization. Elongation of the fatty acyl group by four methylene groups to the natural length (C6) of CDA turned the relaxed regioselectivity into a strict regioselectivity, yielding solely the decapeptide lactone ring, along with decreased hydrolysis of the peptidyl-thioester substrate. This provides evidence for the crucial role of the lipid chain in controlling the regio- and chemoselectivity of TE-mediated macrocyclization. CDA belongs to the group of acidic lipopeptides, which includes the clinically approved antibiotic daptomycin. To evaluate the capability of CDA cyclase for the chemoenzymatic generation of daptomycin, six daptomycin-specific residues were successively incorporated into linear CDA undecapeptidyl-thioesters. All these six substrates were efficiently cyclized by CDA TE. Simultaneous incorporation of all six of these residues into the peptide backbone and elongation of the N-terminus of CDA by two residues finally yielded a daptomycin derivative that lacked only the -methyl group of L-3-methylglutamate. In accordance with acidic lipopeptide antibiotics, the bioactivity of the chemoenzymatic assembled daptomycin analogue is dependent on the presence of calcium ions. To identify calcium-binding sites in the lipo-tridecapeptide chain of the daptomycin analogue, all four acidic residues were successively substituted by either Asn or Gln. Bioactivity studies revealed that only Asp7 and Asp9 are essential for antimicrobial potency. Moreover, these two residues are strictly conserved among all other nonribosomal acidic lipopeptides and the calcium-binding EF-motif of ribosomally assembled calmodulin. The final part of this work is dedicated to the selective detection of peptide cyclization by fluorescence resonance energy transfer (FRET). In this approach, peptide cyclization catalyzed by NRPS-derived TE domains brings the donor Trp and the acceptor Kyn (kynurenine) in sufficiently close proximity to enable efficient FRET. Theses fluorophores were readily incorporated into the peptide backbone by solid-phase peptide chemistry and show excellent spectral overlap between the donor emission and acceptor absorption. Application of this method provided a tool to track TE-mediated peptide cyclization in real-time. Furthermore, picomolar detection limits of cyclopeptides were realized, thereby facilitating kinetic studies of TE-mediated macrocyclization. The general utility of FRET-assisted detection of cyclopeptides was demonstrated for two cyclases, namely tyrocidine (Tyc) TE, and CDA TE. For the latter cyclase, this approach was combined with site-directed affinity labelling, opening the possibility for high-throughput enzymatic screening.
DOI:https://doi.org/10.17192/z2005.0528