Phylogenetic analysis of condensation domains in NRPS sheds light on their functional evolution

摘要

Background Non-ribosomal peptide synthetases (NRPSs) are large multimodular enzymes that synthesize a wide range of biologically active natural peptide compounds, of which many are pharmacologically important. Peptide bond formation is catalyzed by the Condensation (C) domain. Various functional subtypes of the C domain exist: An LC_L domain catalyzes a peptide bond between two L-amino acids, a DC_L domain links an L-amino acid to a growing peptide ending with a D-amino acid, a Starter C domain (first denominated and classified as a separate subtype here) acylates the first amino acid with a β-hydroxy-carboxylic acid (typically a β-hydroxyl fatty acid), and Heterocyclization (Cyc) domains catalyze both peptide bond formation and subsequent cyclization of cysteine, serine or threonine residues. The homologous Epimerization (E) domain flips the chirality of the last amino acid in the growing peptide; Dual E/C domains catalyze both epimerization and condensation. Results In this paper, we report on the reconstruction of the phylogenetic relationship of NRPS C domain subtypes and analyze in detail the sequence motifs of recently discovered subtypes (Dual E/C, DC_L and Starter domains) and their characteristic sequence differences, mutually and in comparison with LC_L domains. Based on their phylogeny and the comparison of their sequence motifs, LC_L and Starter domains appear to be more closely related to each other than to other subtypes, though pronounced differences in some segments of the protein account for the unequal donor substrates (amino vs. β-hydroxy-carboxylic acid). Furthermore, on the basis of phylogeny and the comparison of sequence motifs, we conclude that Dual E/C and DC_L domains share a common ancestor. In the same way, the evolutionary origin of a C domain of unknown function in glycopeptide (GP) NRPSs can be determined to be an LC_L domain. In the case of two GP C domains which are most similar to DC_L but which have LC_L activity, we postulate convergent evolution. Conclusion We systematize all C domain subtypes including the novel Starter C domain. With our results, it will be easier to decide the subtype of unknown C domains as we provide profile Hidden Markov Models (pHMMs) for the sequence motifs as well as for the entire sequences. The determined specificity conferring positions will be helpful for the mutation of one subtype into another, e.g. turning DC_L to LC_L, which can be a useful step for obtaining novel products.