Timing of Epimerization and Condensation Reactions in Nonribosomal Peptide Assembly Lines: Kinetic Analysis of Phenylalanine Activating Elongation Modules of Tyrocidine Synthetase B †

Philipps University of Marburg, Marburg, Hesse, Germany
Biochemistry (Impact Factor: 3.02). 08/2002; 41(29):9184-96. DOI: 10.1021/bi026047+
Source: PubMed


The cyclic decapeptide antibiotic tyrocidine has D-Phe residues at positions 1 and 4, produced during peptide chain growth from L-Phe residues by 50 kDa epimerase (E) domains embedded, respectively, in the initiation module (TycA) and the TycB3 module of the three-subunit (TycABC), 10-module nonribosomal peptide synthetase. While the initiation module clearly epimerizes the aminoacyl thioester Phe1-S-TycA intermediate, the timing of epimerization versus peptide bond condensation at internal E domains has been less well characterized in nonribosomal peptide synthetases. In this study, we use rapid quench techniques to evaluate a three-domain (ATE) and a four-domain version (CATE) of the TycB3 module and a six-domain fragment (ATCATE) of the TycB2(-3) bimodule to measure the ability of the E domain in the TycB3 module to epimerize the aminoacyl thioester Phe-S-TycB3 and the dipeptidyl-S-enzyme (L-Phe-L-Phe-S-TycB3 if L-Phe-D-Phe-S-TycB3). The chiralities of the Phe-S-enzyme and Phe-Phe-S-enzyme species over time were determined by hydrolysis and chiral TLC separations, allowing for the clear conclusion that epimerization in the internal TycB3 module occurs preferentially on the peptidyl-S-enzyme rather than the aminoacyl-S-enzyme, by a factor of about 3000/1. In turn, this imposes constraints on the chiral selectivity of the condensation (C) domains immediately upstream and downstream of E domains. The stereoselectivity of the upstream C domain was shown to be L-selective at both donor and acceptor sites ((L)C(L)) by site-directed mutagenesis studies of an E domain active site residue and using the small-molecule surrogate D-Phe-Pro-L-Phe-N-acetylcysteamine thioester (D-Phe-Pro-L-Phe-SNAC) and D-Phe-Pro-D-Phe-SNAC as donor probes.

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    • "The different core motifs in Condensation domains have first been described by de Crécy-Lagard et al. [28] and recompiled by Marahiel et al. [29] but have never been updated since then. The core motifs of the C domain homologues, Epimerization and Heterocyclization domain are listed in the publication by Marahiel et al. [29] but the sequence motifs of the recently discovered DCL domains [12,30] as well as the Dual E/C [13] domains have never been comprehensively analyzed. Moreover the Starter C domain has not yet been recognized in the literature as a proper separate subtype. "
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    ABSTRACT: 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 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 beta-hydroxy-carboxylic acid (typically a beta-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. 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, 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. beta-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. 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 DCL to LCL, which can be a useful step for obtaining novel products.
    BMC Evolutionary Biology 02/2007; 7(1):78. DOI:10.1186/1471-2148-7-78 · 3.37 Impact Factor
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    • "The presence of D-amino acid residues is a hallmark of nonribosomal peptides. This creates structural diversity, constrains their stereochemical conformation, and resists to proteolysis [2]. Biosynthesis of nonribosomal peptides occurs via the action of catalytic units of NRPS, referred to as modules, in the direction from N-terminal to C-terminal ends as well as by ribosome dependent mechanism. "
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    ABSTRACT: Condensation (C) domains in the nonribosomal peptide synthetases are capable of catalyzing peptide bond formation between two consecutively bound various amino acids. C-domains coincide in frequency with the number of peptide bonds in the product peptide. In this study, a phylogenetic approach was used to investigate structural diversity of bacterial C-domains. Phylogenetic trees show that the C-domains are clustered into three functional groups according to the types of substrate donor molecules. They are l-peptidyl donors, d-peptidyl donors, and N-acyl donors. The fact that C-domain structure is not subject to optical configuration of amino acid acceptor molecules supports an idea that the conversion from l to d-form of incorporating amino acid acceptor occurs during or after peptide bond formation. l-peptidyl donors and d-peptidyl donors are suggested to separate before separating the lineage of Gram-positive and Gram-negative bacteria in the evolution process.
    FEMS Microbiology Letters 12/2005; 252(1):143-51. DOI:10.1016/j.femsle.2005.08.041 · 2.12 Impact Factor
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    • "amino acyl instead of peptidyl moieties, or peptidyl chains with different size and amino acid composition (Belshaw et al., 1999; Doekel & Marahiel, 2000; Linne et al., 2003; Marshall et al., 2001), but no kinetic data are yet available to quantify the catalytic efficiency of these condensations. The stereochemistry of the C-terminal amino acid of the peptidyl chain appears, however, to be an important element in substrate recognition , as observed for the acceptor site (Ehmann et al., 2000; Luo et al., 2002). It is interesting that C domains appear to be more selective at their acceptor site than at their donor site, because this means that the C domain exhibits greater selectivity towards the amino acid activated by the A domain within the same module compared with those amino acids activated by A domains in upstream modules. "
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    ABSTRACT: Nonribosomal peptide synthetases (NRPSs) are giant multi-domain enzymes that catalyse the biosynthesis of many commercially important peptides produced by bacteria and fungi. Several studies over the last decade have shown that many of the individual domains within NRPSs exhibit significant substrate selectivity, which impacts on our ability to engineer NRPSs to produce new bioactive microbial peptides. Adenylation domains appear to be the primary determinants of substrate selectivity in NRPSs. Much progress has been made towards an empirical understanding of substrate selection by these domains over the last 5 years, but the molecular basis of substrate selectivity in these domains is not yet well understood. Perhaps surprisingly, condensation domains have also been reported to exhibit moderate to high substrate selectivity, although the generality of this observation and its potential impact on engineered biosynthesis experiments has yet to be fully elucidated. The situation is less clear for the thioesterase domains, which seem in certain cases to be dedicated to the hydrolysis/cyclization of their natural substrate, whereas in other cases they are largely permissive.
    Microbiology 07/2004; 150(Pt 6):1629-36. DOI:10.1099/mic.0.26837-0 · 2.56 Impact Factor
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