Chemoenzymatic and Template‐Directed Synthesis of Bioactive Macrocyclic Peptides

Abstract

SummaryNonribosomal 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 Thr 2 and Ser 1 residues onto the C-terminal Trp 11 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 Thr 2 or Ser 1 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 Lconfigured Ser 1 and Thr 2 for cyclization. Elongation of the fatty acyl group by four methylene groups to the natural length (C 6 ) 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 β-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 residue...

Iron Transport and Signaling in Pseudomonads

“…Nonribosomal peptide synthesis occurs via a multiple-carrier thiotemplate mechanism on large enzymes called nonribosomal peptide synthetases (NRPSs) (reviewed in Ref. [60]). NRPSs are organized into sets of iterative catalytic units called modules, with each module responsible for the incorporation of a specific amino acid or other building block into the product.…”

Nonribosomal Peptides

“…This method works fairly well; however, it seems in later studies that the acylation potential is of greater importance than the similarity to the natural situation and a variety of peptidyl-thioesters with better leaving groups than SNAc was tested. The fastest turnover rates were found when thiophenol-esters were used (30,34). Thiophenol has several advantages: It does not have any functional groups other than the thiol group, it is inexpensive, and it can easily be separated from the product.…”

“…A very powerful method for producing novel antibiotics is the chemoenzymatic approach (30). The idea behind this strategy is to leave out the enzymatic buildup of the linear peptide scaffolds and replace it by solid-phase peptide synthesis ( Fig.…”

Cloning and molecular characterization of the gene encoding the Aureobasidin A biosynthesis complex in Aureobasidium pullulans BP-1938

“…As a whole, the mechanism readily explains the specific characteristics associated with many cyclic peptides, such as the inclusion of non-proteinogenic and N-methylated amino acids, ester bonds, D-amino acids, and also the final cyclization of the molecules. Since each adenylation domain is specific for a certain amino acid (or modification thereof), the sequential arrangement of the domains in a type A NRPS complex (Grünewald and Marahiel, 2006) does, in itself, determine the sequence and structure of the cyclic peptide produced. From this follows that if the sequence, or order, of the domains in the enzyme complex is changed, the amino acid sequence, or position of modifications, in the cyclic peptide synthesized by the complex should also change.…”