Structural elements of an NRPS cyclization domain and its intermodule docking domain

Dowling, Daniel; Kung, Yan; Croft, Anna K.; Taghizadeh, Koli; Kelly, Walton R.; Walsh, Christopher T.; Drennan, Catherine L.

doi:10.1073/pnas.1608615113

Cited by 71 publications

(112 citation statements)

References 52 publications

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“…[38] Thespontaneous conversion of 1 is predicted to occur via at etrahedral hemiorthoamide intermediate formed by attack of the amine on the ester (Figure 6a). In RiPP [24] and NRP [29][30][31][32] systems,o xazoline biosyn-thesis also proceeds via this hemiorthoamide intermediate (phosphorylated in RiPP) but it is formed by the attack of hydroxyl upon an amide (Figure 1b).…”

Section: Resultsmentioning

confidence: 99%

“…Unconjugated oxazoles are mostly produced by ribosomally synthesized post‐translationally modified peptides (RiPPs) synthesis and by non‐ribosomal peptides (NRPs) synthesis . Oxazoles in both families derive from nucleophilic attack of the hydroxyl group of Ser(Thr) upon amide bonds through a common hemiorthoamide intermediate (Figure b), but via distinct enzymatic routes . Two polyketide synthase–NRPS hybrid natural products, oxazolomycin and conglobatin, have been proposed to utilize an N‐type ATP pyrophosphohydrolase to form oxazoles .…”

Section: Introductionmentioning

confidence: 99%

“…[20,21] Oxazoles in both families derive from nucleophilic attack of the hydroxyl group of Ser(Thr) upon amide bonds [20,22] through ac ommon hemiorthoamide intermediate ( Figure 1b), but via distinct enzymatic routes. [23][24][25][26][27][28][29][30][31][32][33][34] Tw o polyketide synthase-NRPS hybrid natural products,o xazolomycin and conglobatin, have been proposed to utilize an Ntype ATPpyrophosphohydrolase to form oxazoles. [35,36] None of these enzymes have been reported to catalyze the formation of benzoxazole and such activity would seem unlikely based on their known substrates.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

The Biosynthesis of the Benzoxazole in Nataxazole Proceeds via an Unstable Ester and has Synthetic Utility

et al. 2020

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Heterocycles, a class of molecules that includes oxazoles, constitute one of the most common building blocks in current pharmaceuticals and are common in medicinally important natural products. The antitumor natural product nataxazole is a model for a large class of benzoxazole‐containing molecules that are made by a pathway that is not characterized. We report structural, biochemical, and chemical evidence that benzoxazole biosynthesis proceeds through an ester generated by an ATP‐dependent adenylating enzyme. The ester rearranges via a tetrahedral hemiorthoamide to yield an amide, which is a shunt product and not, as previously thought, an intermediate in the pathway. A second zinc‐dependent enzyme catalyzes the formation of hemiorthoamide from the ester but, by shuttling protons, the enzyme eliminates water, a reverse hydrolysis reaction, to yield the benzoxazole and avoids the amide. These insights have allowed us to harness the pathway to synthesize a series of novel halogenated benzoxazoles.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Biosynthesis of the Benzoxazole in Nataxazole Proceeds via an Unstable Ester and has Synthetic Utility

et al. 2020

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“…During the review process for this study, a structure and mutational analysis of the heterocyclization domain of epothilone synthase (EpoB-Cy) was reported by Dowling et al (54). The structures of EpoB-Cy and BmdB-Cy2 are similar.…”

Section: Insight Into Catalysis and Model Of The Cyclodehydration Intmentioning

confidence: 87%

Structural and mutational analysis of the nonribosomal peptide synthetase heterocyclization domain provides insight into catalysis

Bloudoff

Fage

Marahiel

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

115

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Nonribosomal peptide synthetases (NRPSs) are a family of multidomain, multimodule enzymes that synthesize structurally and functionally diverse peptides, many of which are of great therapeutic or commercial value. The central chemical step of peptide synthesis is amide bond formation, which is typically catalyzed by the condensation (C) domain. In many NRPS modules, the C domain is replaced by the heterocyclization (Cy) domain, a homologous domain that performs two consecutive reactions by using hitherto unknown catalytic mechanisms. It first catalyzes amide bond formation, and then the intramolecular cyclodehydration between a Cys, Ser, or Thr side chain and the backbone carbonyl carbon to form a thiazoline, oxazoline, or methyloxazoline ring. The rings are important for the form and function of the peptide product. We present the crystal structure of an NRPS Cy domain, Cy2 of bacillamide synthetase, at a resolution of 2.3 Å. Despite sharing the same fold, the active sites of C and Cy domains have important differences. The structure allowed us to probe the roles of active-site residues by using mutational analyses in a peptide synthesis assay with intact bacillamide synthetase. The drastically different effects of these mutants, interpreted by using our structural and bioinformatic results, provide insight into the catalytic mechanisms of the Cy domain and implicate a previously unexamined Asp-Thr dyad in catalysis of the cyclodehydration reaction.nonribosomal peptide synthetase | heterocyclization domain | X-ray crystallography | bacillamide | mutational analysis

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“…While for PKS three different types of DD pairs have been described structurally so far, two structures for N DDs without a bound C DD have been solved for NRPS/PKS hybrid systems. 7–12…”

Section: Introductionmentioning

confidence: 99%

Structure-based redesign of docking domain interactions modulates the product spectrum of a rhabdopeptide-synthesizing NRPS

Hacker

Cai

Kegler

et al. 2018

Preprint

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18Several peptides in clinical use are derived from non-ribosomal peptide synthetases 19 (NRPS). In these systems multiple NRPS subunits interact with each other in a 20 specific linear order mediated by docking domains (DDs) to synthesize well-defined 21 peptide products. In contrast to these classical NRPSs, the subunits of 22 23 resulting in libraries of peptide products. In order to define the structural and 24 thermodynamic basis for their unusual interaction patterns, we determined the 25 structures of all N-terminal DDs ( N DDs) as well as of an N DD-C DD complex and 26 characterized all putative DD interactions thermodynamically for one such system. 27Key amino acid residues for DD interactions were identified that upon their exchange 28 not only changed the DD affinity but also resulted in rationally predictable changes in 29 peptide production. A simple set of 'recognition rules' for DD interactions was 30 identified that also operates in other megasynthase complexes. 31 32 42 C-terminal docking domains (DDs). Stachelhaus and coworkers have demonstrated 43 that a matching pair of DDs or COM (communication-mediating) domains at the N-44 terminus of peptidyl-donating NRPS ( N DD) and the C-terminus of accepting NRPS 45 ( C DD) play a decisive role in defining the order of interactions between subunits in 46 vitro and in vivo by swapping COM domains. 3 They also constructed a "universal 47 3 COM system" in vitro by comparison of COM domains in tyrocidin A and surfactin-like 48 NRPSs, which led to enzyme crosstalk between different biosynthetic systems that 49 promoted the combinatorial biosynthesis of different peptides. 4,5 However, the 50 structures of these DDs have not been elucidated so far but interactions between 51 DDs have been mapped based on photo crosslinking experiments. 6 More information 52 on DDs is available for polyketide synthase (PKS) or PKS/NRPS hybrid systems. 53 While for PKS three different types of DD pairs have been described structurally so 54 far, two structures for N DDs without a bound C DD have been solved for NRPS/PKS 55 hybrid systems. 7-12 56 Classic NRPSs are multimodular -each protein subunit normally consists of the 57 processing modules for multiple amino acids arranged in a linear fashion. 58 Furthermore, the different protein subunits interact with each other in a strictly 59 defined linear order yielding a single peptide product with a sequence that faithfully 60 reproduces the linear order of the processing modules along the protein chains. A 61 novel class of monomodular NRPSs involved in the production of libraries of 62 rhabdopeptide/xenortide peptides (RXPs) has been recently described from 63 entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus. 13 The 64RXP product spectrum in a single strain differs mainly in peptide length and 65 sequence, which requires an iterative use of certain NRPS modules and is 66 dependent on protein stoichiometry between NRPS modules acting in elongation and 67 termination ( Supplementary Figure 1). This suggests th...

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Structural elements of an NRPS cyclization domain and its intermodule docking domain

Cited by 71 publications

References 52 publications

The Biosynthesis of the Benzoxazole in Nataxazole Proceeds via an Unstable Ester and has Synthetic Utility

The Biosynthesis of the Benzoxazole in Nataxazole Proceeds via an Unstable Ester and has Synthetic Utility

Structural and mutational analysis of the nonribosomal peptide synthetase heterocyclization domain provides insight into catalysis

Structure-based redesign of docking domain interactions modulates the product spectrum of a rhabdopeptide-synthesizing NRPS

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