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

Abstract

BackgroundNon-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 LCL domain catalyzes a peptide bond between two L-amino acids, a DCL 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.ResultsIn 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, DCL and Starter domains) and their characteristic sequence differences, mutually and in comparison with LCL domains. Based on their phylogeny and the comparison of their sequence motifs, LCL 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 DCL 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 LCL domain. In the case of two GP C domains which are most similar to DCL but which have LCL activity, we postulate convergent evolution.ConclusionWe 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 DCL to LCL, which can be a useful step for obtaining novel products.

Deep sequencing of non-ribosomal peptide synthetases and polyketide synthases from the microbiomes of Australian marine sponges

“…The C-terminus binding pocket, considered the acceptor site, exhibits specific selectivity for the activated substrate, which is conferred within the amino-acid sequence. This allows for the discrimination between activated L-amino, D-amino or N-acyl substrates (Roongsawang et al, 2005;Rausch et al, 2007). Within this study, both the TEFAP and mWGS approaches were in agreement as to the presence of only LCL domains within Scopalina sp., C. concentrica and C. coralliophila.…”

“…Following screening, the TEFAP reads and mWGS (open reading frames) were combined. Previous phylogenetic analyses of KS and C domains have indicated that these domains typically exhibit at least 5% amino-acid dissimilarity (Moffitt and Neilan, 2003;Roongsawang et al, 2005;Rausch et al, 2007). Previous TEFAP methods have reflected this amino-acid dissimilarity by clustering nucleotides at a 90% nucleotide similarity cutoff (Varaljay et al, 2010).…”

“…Reference sequences were selected on the basis of either belonging to known, characterised biosynthetic pathways or due to their previously established phylogenetic distribution (Moffitt and Neilan, 2003;Roongsawang et al, 2005;Fieseler et al, 2007;Rausch et al, 2007). As a result, the C domain alignment contained 162 reference sequences while the KS domain alignment had 190 reference sequences.…”

“…The KS and condensation (C) domains, that catalyse the condensation of two activated subunits, contain motifs that are conserved at an amino-acid level (Stachelhaus et al, 1998;Walsh and Fischbach, 2010). Between these motifs, variation is observed, typically within catalytic centres, giving rise to domains with functional specificity, reflecting the nature of the subunits being condensed (Moffitt and Neilan, 2003;Rausch et al, 2007), the arrangement of other domains within the module and the expected product (Moffitt and Neilan, 2003;Piel et al, 2004a;Schmitt et al, 2008). The conserved nature for part of these domains ensures their suitability as targets for amplification using the PCR.…”

Fungal cyclooligomerdepsipeptides: From classical biochemistry to combinatorial biosynthesis

“…NRPS C domains were shown to form several phylogenetic clades corresponding to functional subtypes. 102 Although the C 2 domains of CODS catalyze a condensation between substrates of D and an L configuration (i.e. they are formally D C L domains), their core motifs are nevertheless more similar to domains of the L C L subtype.…”