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. 2004 Nov 2;101(44):15585-90.
doi: 10.1073/pnas.0404932101. Epub 2004 Oct 21.

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

Martin Hahn  1 , Torsten Stachelhaus
Affiliations

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

Martin Hahn et al. Proc Natl Acad Sci U S A. .

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 protein-protein 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.

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Figures

Fig. 1.
Fig. 1.
The Tyc biosynthetic system. The enzymatic assembly line of the cyclic decapeptide antibiotic TycA (C) consists of three NRPSs: TycA (124 kDa), TycB (405 kDa), and TycC (724 kDa), which are encoded by the polycistronic genes tycABC (A). The NRPSs are composed of one, three, and six modules, respectively, each one responsible for the incorporation of one monomeric amino acid (B). In accordance with the colinearity principle, protein–protein communication has to occur between TycA and TycB1, as well as between TycB3 and TycC1. (D) Artificial dimodular NRPS systems TycA/TycB1 and TycB3/TycC1 had been constructed, which catalyze in vitro synthesis of the dipeptide products DPhe-LPro-DKP and DPhe-LAsn, respectively.
Fig. 2.
Fig. 2.
Sequence comparison of the junction among seven partner NRPSs derived from four different biosynthetic systems [Tyc, Srf, gramicidin S (Grs), and lichenysin(Lic)]. Only the most C-terminal residues of the donor module and the most N-terminal residues of corresponding acceptor module are shown. Invariant amino acid residues are shaded in black, and residues that are conserved in the majority of the COM domains are shaded in gray. Localization of the proposed C- and N-terminal COM domains is indicated.
Fig. 3.
Fig. 3.
Elongation activity of C-terminal TycA deletion mutants. (A) Elongation activity was determined with the partner elongation module TycB1 by measuring the amount of enzyme-bound, acid-stable label. (B) DKP product formation was monitored after organic extraction by the accumulation of radiolabeled D[14C]Phe-LPro-DKP in the organic layer. For the sake of clarity, mutant systems TycAΔ12–23/TycB1 were omitted from the presentation but gave essentially the same patterns as the control reaction.
Fig. 4.
Fig. 4.
Time-dependent formation of DPhe-LPro-DKP. Quantitative HPLC analysis was carried out by using the partner NRPS system TycA/TycB1 with all possible combinations of COM domains as well as the nonpartner system TycB3(A)/TycB1.
Fig. 5.
Fig. 5.
Crosstalk between the Tyc and Srf biosynthetic systems. The crosstalk was investigated for the nonpartner NRPS systems TycA/SrfAC, TycB3(A)/SrfAC, TycA(B3)/SrfAC, and TycB3/SrfAC. HPLC/MS revealed, only for the first two systems, the formation of the expected DPhe-LLeu dipeptide product (retention time, 30.8 min; [M+H]+ = 279.1 m/z).
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References

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