Nonribosomal peptides for iron acquisition: pyochelin biosynthesis as a case study

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Highlights

  • •

    Domain interactions and alternations of full-module and cross-module NRPSs are discussed.

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    Biodiversity of NRPS products is provided by accessory and tailoring enzymes.

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    Stuffed tailoring domains are an understudied class of NRPS biosynthesis.

  • •

    Stuffed epimerase and methyltransferase domains share architecture and substrates.

Microbes synthesize small, iron-chelating molecules known as siderophores to acquire iron from the environment. One way siderophores are generated is by nonribosomal peptide synthetases (NRPSs). The bioactive peptides generated by NRPS enzymes have unique chemical features, which are incorporated by accessory and tailoring domains or proteins. The first part of this review summarizes recent progress in NRPS structural biology. The second part uses the biosynthesis of pyochelin, a siderophore from Pseudomonas aeruginosa, as a case study to examine enzymatic methods for generating the observed diversity in NRPS-derived natural products.

Section snippets

Iron uptake: siderophores

Iron is an essential nutrient that is needed by microbes to perform critical biological processes necessary for survival [1]. Due to the paucity of free iron in aerobic environments, microbes have developed intricate systems to acquire iron from their surroundings [1]. One such system is the biosynthesis of small molecules known as siderophores that have a high affinity for ferric iron [2]. When iron availability is low, microbes synthesize and secrete siderophores, and selectively reimport the

Nonribosomal peptide synthetases (NRPS): chemical logic of peptide chain formation

NRPS enzymes are large multidomain and multifunctional enzymes that display a chemical logic in which each module is responsible for the addition of a single amino acid to a growing peptide chain, including non-proteinogenic amino acids and hydroxy acids. An NRPS module consists of a condensation (C) domain, an adenylation (A) domain, and a peptidyl carrier protein (P) domain, except for the first initiation module, which lacks a condensation domain. The P-domain acts as a tethering system for

Nonribosomal peptide synthetases (NRPS): structural biology of an assembly line

The C-domain, A-domain, and P-domain have been well-studied, with both structural and kinetic characterizations [9, 10, 11, 12, 13]. The first structure elucidating the organization of a full NRPS module was the termination module (C-A-P-T) of surfactin biosynthesis, SrfA-C, from Bacillus subtilis (Figure 1b) [14]. Crystals of the full module were obtained after mutating the serine of the P-domain that is post-translationally modified with Ppant to alanine. The A-domain contains two subdomains,

Pyochelin from Pseudomonas aeruginosa

The chemical logic and structural biology of NRPS modules is key for understanding how the peptide bonds are made by these nanomachines. However, the bioactive peptides produced by these enzymes are far more diverse than the 20 amino acids found in proteins. The diversity is incorporated by accessory enzymes that produce unusual amino and hydroxy acid substrates for adenylation enzymes or by tailoring enzymes that alter amino acids already incorporated into the growing chain. Here, we use the

Accessory enzymes in pyochelin biosynthesis: PchA and PchB

PchA, isochorismate synthase. PchA catalyzes the first step in pyochelin biosynthesis, isomerizing chorismate to isochorismate (Figure 3a) [29, 30]. PchA is part of the menaquinone, siderophore, and tryptophan (MST) family of enzymes that are Mg2+-dependent chorismate utilizing enzymes [34, 35, 36]. Although the structure of PchA remains unsolved, the structure is hypothesized to be homologous to the isochorismate synthases, EntC [37, 38] and MenF [35] (both E. coli enzymes) and the salicylate

Stuffed tailoring domains in pyochelin biosynthesis: PchE and PchF

Most commonly, tailoring domains are incorporated into an NRPS module as an independent domain following the P-domain. However, in some cases tailoring domains are inserted within the A-domain, considered ‘interrupted’ adenylation domains [52]. The tailoring domains of both PchE and PchF are stuffed into interrupted adenylation domains between the core sequence motifs, A8 and A9, of the Asub-domain (Figure 4a) [33, 52]. The adenylation-tailoring didomains lack structural characterization and

Stand-alone tailoring domain: PchG

The terminal cyclized cysteine must be reduced to a thiazolidine while tethered to the Ppant tail on PchF, prior to final methylation and release of the mature pyochelin by the thioesterase domain (Figure 4f) [27]. PchG, a stand-alone tailoring enzyme, is proposed to perform the reduction by proton donation from a general acid and subsequent hydride transfer from NADPH. Structures of a functionally homologous enzyme of yersiniabactin biosynthesis [67], Irp3, have been determined: an apo-form,

Conclusion

Siderophores are small, iron-chelating molecules generated by NRPS systems. NRPS structures have provided insight into how these nanomachines form peptide bonds outside of the ribosome. Recently, several full-module structures have elucidated the different domain interactions and domain alternations that occur during peptide assembly. Additional full-module and cross-module structures are necessary to understand domain interactions between modules. While NRPS enzymes generate peptide assembly,

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

This publication was made possible by funds from The National Science Foundation (CHE-1403293). T.A.R. was supported by the National Institutes of Health Graduate Training Program in the Dynamic Aspects of Chemical Biology (T32 GM008545). Special thanks to Dr. Catherine Shelton and Jeffrey McFarlane for critically reading the manuscript.

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