New Structural Data Reveal the Motion of Carrier Proteins in Nonribosomal Peptide Synthesis

Kittilä, Tiia; Mollo, Aurelio; Charkoudian, Louise K.; Cryle, Max J.

doi:10.1002/anie.201602614

Cited by 51 publications

(45 citation statements)

References 53 publications

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“…Numerous reviews exist, 15, 16, 22 including several that are quite recent, 48-55 that detail what is known about the structures of both catalytic domains and larger multidomain and modular proteins. Indeed, this is an exciting time for the structural studies of NRPSs as many structures of multidomain proteins were determined in 2016.…”

Section: The Biochemistry and Structural Biology Of The Nonribosommentioning

confidence: 99%

Nonribosomal peptide synthetase biosynthetic clusters of ESKAPE pathogens

Gulick

2017

Nat. Prod. Rep.

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Overview Natural products are important secondary metabolites produced by bacterial and fungal species that play important roles in cellular growth and signaling, nutrient acquisition, intra- and interspecies communication, and virulence. A subset of natural products is produced by nonribosomal peptide synthetases (NRPSs), a family of large, modular enzymes that function in an assembly line fashion. Because of the pharmaceutical activity of many NRPS products, much effort has gone into the exploration of their biosynthetic pathways and the diverse products they make. Many interesting NRPS pathways have been identified and characterized from both terrestrial and marine bacterial sources. Recently, several NRPS pathways in human commensal bacterial species have been identified that produce molecules with antibiotic activity, suggesting another source of interesting NRPS pathways may be the commensal and pathogenic bacteria that live on the human body. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) have been identified as a significant cause of human bacterial infections that are frequently multidrug resistant. The emerging resistance profile of these organisms has prompted calls from multiple international agencies to identify novel antibacterial targets and develop new approaches to treat infections from ESKAPE pathogens. Each of these species contains several NRPS biosynthetic gene clusters. While some have been well characterized and produce known natural products with important biological roles in microbial physiology, others have yet to be investigated. This review catalogs the NRPS pathways of ESKAPE pathogens. The exploration of novel NRPS products may lead to a better understanding of the chemical communication used by human pathogens and potentially to the discovery of novel therapeutic approaches.

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Section: The Biochemistry and Structural Biology Of The Nonribosommentioning

confidence: 99%

Nonribosomal peptide synthetase biosynthetic clusters of ESKAPE pathogens

Gulick

2017

Nat. Prod. Rep.

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“…The biosynthesis of GPAs is based around the initial synthesis of the linear heptapeptide by a type-I non-ribosomal peptide synthetase (NRPS) [5–6] and its subsequent modification by cytochrome P450 monooxygenases [7–9], which install the crosslinks that provide the unique structure and hence activity of the GPAs (Fig. 1) [4].…”

Section: Introductionmentioning

confidence: 99%

Biochemical and structural characterisation of the second oxidative crosslinking step during the biosynthesis of the glycopeptide antibiotic A47934

Ulrich¹,

Brieke²,

Cryle³

2016

Beilstein J. Org. Chem.

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The chemical complexity and biological activity of the glycopeptide antibiotics (GPAs) stems from their unique crosslinked structure, which is generated by the actions of cytochrome P450 (Oxy) enzymes that affect the crosslinking of aromatic side chains of amino acid residues contained within the GPA heptapeptide precursor. Given the crucial role peptide cyclisation plays in GPA activity, the characterisation of this process is of great importance in understanding the biosynthesis of these important antibiotics. Here, we report the cyclisation activity and crystal structure of StaF, the D-O-E ring forming Oxy enzyme from A47934 biosynthesis. Our results show that the specificity of StaF is reduced when compared to Oxy enzymes catalysing C-O-D ring formation and that this activity relies on interactions with the non-ribosomal peptide synthetase via the X-domain. Despite the interaction of StaF with the A47934 X-domain being weaker than for the preceding Oxy enzyme StaH, StaF retains higher levels of in vitro activity: we postulate that this is due to the ability of the StaF/X-domain complex to allow substrate reorganisation after initial complex formation has occurred. These results highlight the importance of testing different peptide/protein carrier constructs for in vitro GPA cyclisation assays and show that different Oxy homologues can display significantly different catalytic propensities despite their overall similarities.

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“…6 In all these cases, however, the assessment of enzymatic activity is complicated by the necessity of peptide intermediates to be bound to peptidyl carrier protein (PCP)-domains, which serve as an attachment point for all amino acid and peptide intermediates during NRPS biosynthesis. 1,2,8 The ability to enzymatically load coenzyme A (CoA) substrates onto PCP domains using the promiscuous phosphopantetheinyl transferase Sfp has made a vital contribution to overcome this problem, as it allows biosynthetic steps to be interrogated without complete reconstitution of the NRPS. 1,8 It does, however, require effective methods for the generation of peptide thioester CoA substrates to be able to undertake these experiments.…”

mentioning

confidence: 99%

“…1,2,8 The ability to enzymatically load coenzyme A (CoA) substrates onto PCP domains using the promiscuous phosphopantetheinyl transferase Sfp has made a vital contribution to overcome this problem, as it allows biosynthetic steps to be interrogated without complete reconstitution of the NRPS. 1,8 It does, however, require effective methods for the generation of peptide thioester CoA substrates to be able to undertake these experiments. However, the diversity of amino acid monomers utilised by NRPS machineries can now present serious problems for such syntheses, with no better example found than the phenylglycine residues that form the majority of residues in the peptide core of GPAs such as teicoplanin.…”

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confidence: 99%

A route to diastereomerically pure phenylglycine thioester peptides: crucial intermediates for investigating glycopeptide antibiotic biosynthesis

et al. 2018

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abc Non-ribosomal peptides contain an array of amino acid building blocks that can present challenges for the synthesis of important intermediates. Here, we report the synthesis of glycopeptide antibiotic (GPA) thioester peptides that retains the crucial stereochemical purity of the terminal phenylglycine residue, which we show is essential for the enzymatic GPA cyclisation cascade.Non-ribosomal peptide synthesis is central to the biosynthesis of many compounds of medical importance, including a large number of antibiotics. [1][2][3][4] Due to the modular structure of nonribosomal peptide synthetases (NRPSs) -comprising repeating domains performing specific catalytic functions -and their non-dependence on the ribosome, NRPS assembly lines are able to synthesise peptides from a wide range of amino acids. 1,3This feature, combined with the extensive incorporation of (D)-amino acids and large range of further structural modifications, contribute to the extensive diversity of natural NRPS-peptides. 1,2Given the complexity of many of these compounds that are of medical interest -such as the glycopeptide antibiotics ( Fig. 1), with vancomycin and teicoplanin as representative members -there is a need to characterise NRPS biosynthesis in order to explore the possibilities for compound development through redesigning the corresponding biosynthetic machinery. [1][2][3][4] In this regard, GPAs are perfect examples: total synthesis is highly challenging, meaning that all GPAs in clinical use stem from bacterial production in vivo and modified GPAs generated via total synthesis are not always accessible for the use in clinics. 4,5 The main reasons for this synthetic complexity are the high content of racemisation-prone phenylglycine residues as well as the highly crosslinked structure of GPAs (vancomycin:, which is formed by cytochrome P450 (Oxy) enzymes that interact with the unique NRPS domain found in GPA biosynthesis, the X-domain. 4,6,7 Fig. 1 GPA cyclisation in vitro is enabled by diastereomerically pure phenylglycine thioester peptides: teicoplanin seq. 5: R 1 = 4-Hpg; R 2 = Cl; R 3 = 3,5-Dpg; pekiskomycin seq. 6: R 1 = CH 3 ; R 2 = H; R 3 = Glu; actinoidin seq. 7: R 1 = 4-Hpg; R 2 = H; R 3 = Phe; vancomycin seq. 8: R 1 = Leu; R 2 = Cl; R 3 = Asn. PCP -peptidyl carrier protein domain, X -Oxy recruitment domain, A tei -OxyA from the teicoplanin system, B van -OxyB from the vancomycin system.

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New Structural Data Reveal the Motion of Carrier Proteins in Nonribosomal Peptide Synthesis

Cited by 51 publications

References 53 publications

Nonribosomal peptide synthetase biosynthetic clusters of ESKAPE pathogens

Nonribosomal peptide synthetase biosynthetic clusters of ESKAPE pathogens

Biochemical and structural characterisation of the second oxidative crosslinking step during the biosynthesis of the glycopeptide antibiotic A47934

A route to diastereomerically pure phenylglycine thioester peptides: crucial intermediates for investigating glycopeptide antibiotic biosynthesis

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