Selective interaction between nonribosomal peptide synthetases is facilitated by short communication-mediating domains

Abstract

Nonribosomal peptide synthetases (NRPSs) catalyze the formation of structurally diverse and biologically important peptides. Given their modular organization, NRPSs provide an enormous potential for biocombinatorial approaches to generate novel bioactive compounds. Crucial for the exploitation of this potential is a profound knowledge of the intermolecular communication between partner NRPSs. The overall goal of this study was to understand the basis of protein-protein communication that facilitates the selective interaction in these multienzyme complexes. On this account, we studied the relevance of short regions at the termini of the NRPSs tyrocidine (Tyc) synthetases TycA, TycB, and TycC, constituting the Tyc biosynthetic template. In vitro and in vivo investigations of C-terminal deletion mutants of the initiation module TycA provided evidence for the existence and impact of short communication-mediating (COM) domains. Their decisive role in proteinprotein recognition was subsequently proven by means of COM domain-swapping experiments. Substitution of the terminal COM domains between the donor modules TycA and TycB3, as well as between the acceptor modules TycB1 and TycC1, clearly demonstrated that matching pairs of COM domains are both necessary and sufficient for the establishment of communication between partner NRPSs in trans. These results corroborated the generality of COM domains, which were subsequently exploited to induce crosstalk, even between NRPSs derived from different biosynthetic systems. In conclusion, COM domains represent interesting tools for biocombinatorial approaches, which, for example, could be used for the generation of innovative natural product derivatives. N onribosomal peptide synthetases (NRPSs) are large multidomain enzymes responsible for the biosynthesis of many pharmacologically important bioactive compounds of great structural diversity (1-3). Prominent examples are the antibiotics penicillin, vancomycin, and actinomycin D, the immunosuppressant cyclosporine A, the siderophore enterobactin, and the antitumor drug bleomycin. As illustrated in Fig. 1 for the biosynthesis of the nonribosomal peptide (NRP) tyrocidine, NRPSs are organized into distinct modules, each of them responsible for the incorporation of one amino acid into the nascent peptide chain. A module can be further subdivided into catalytic domains, which are responsible for the coordinated recognition and activation [adenylation (A) domain] (4), covalent binding and transfer [peptidyl carrier protein (PCP) domain] (5), and incorporation [condensation (C) domain] of a certain substrate amino acid into the peptide chain (6). In addition to these so-called core domains, optional domains catalyze the modification of incorporated residues, i.e., by epimerization (E) or N-methylation (MT) domains (7). Product release is normally effected by a thioesterase (Te) domain, catalyzing the formation of linear, cyclic, or branched cyclic products, representative for the class of NRPs (8).Because of the modular organization of...

Harnessing the potential of communication-mediating domains for the biocombinatorial synthesis of nonribosomal peptides

“…In multienzymatic NRPS complexes, which actually represent the vast majority of known NRP assembly lines, synthesis also requires the proper communication between partner enzymes and the prevention of unselective interactions between nonpartner enzymes. In the latter case, the necessary selectivity is provided by the interplay of COM D and COM A domains, located at the termini of the corresponding NRPSs (7). Without the selectivity provided by different sets of compatible COM domains, the enzymes of a NRPS complex would form random biosynthetic templates (Fig.…”

“…Construction of most expression plasmids used in this study was described in ref. 7. Plasmid pTrcHis-tycA::COM D ⌬E (domain organization A-PCP-COM D TycA ) is a derivative of pTrcHis-TycA and was obtained by inverse PCR using the oligonucleotides (restriction sites underlined) 5Ј-tycA::COM D ⌬E (5Ј-AAA AGA TCT GAG CGA ACG CCC AGC G-3Ј) and 3Ј-tycA::COM D ⌬E (5Ј-TTT AGA TCT GCT CTT GAC AAA AAG AGC AAC C-3Ј).…”

“…For example, the acyl-S-Ppant substrate of an E domain has to be presented by an designed PCP, whose primary structure is clearly distinct from a ''regular'' PCP, which interacts only with A and C domains (8). The same could also be true for the physical linkage between E and COM D domains, especially because it has been recognized that (i) the C-terminal half (E-COM D TycA ) of TycA (A-T-E-COM D TycA ) acts as an architectural bridge toward TycB1 (10), and (ii) the TycA main body (A-T-E) might contribute to the productive interaction between both partner enzymes (7).…”

“…1). In our quest to decipher the molecular basis for the selective interaction between NRPSs, we recently discovered and verified the decisive role of communication-mediating (COM) domains (7). According to this study, a donor COM domain (COM D ) located at the C terminus of an aminoacyl-or peptidyl-donating NRPS and an acceptor COM domain (COM A ) located at the N terminus of the accepting partner NRPS form a matching (compatible) set, required for the proper intermolecular interaction between adjacent modules (i.e., TycA͞TycB1 and TycB3͞TycC1).…”

Combinatorial biosynthesis of novel antibiotics related to daptomycin

“…Several amino acid residues in the interpeptide docking sites were identified as important for maintaining or preventing functional interactions of NRPS subunits (19,20). In an in vitro approach, rationally designed cyclic peptides and antibiotics were synthesized by using thioesterase-catalyzed chemoenzymatic biosynthesis (21, 22).…”