Piecing together nonribosomal peptide synthesis
Introduction
Nonribosomal peptide synthetases (NRPSs) are a family of microbial megaenzymes that produce natural products that are useful to society as therapeutics (antibiotics, antivirals, antitumours, and immunosuppressants) and green chemicals (agricultural agents, emulsifiers, siderophores, and research tools) (Figure 1a) [3]. NRPSs typically synthesize their products through amide bond formation between aminoacyl (or other acyl) monomers. Their architecture is dissimilar to the more famous peptide maker. Whereas ribosomes use the same active sites for each amino acid added to the ribosomal peptide, NRPSs typically employ a dedicated set of enzyme domains for each amino acid added to the nonribosomal peptide. This set of domains is termed a module, and the synthetic strategy dictates that, normally, the number and specificity of the modules correspond to the length and sequence of amino acids in the peptide product. NRPSs can consist of a single polypeptide of between 2 and 18 modules, with a mass of ∼220 kDa to 2.2 MDa, or be split over multiple proteins that assemble non-covalently (Figure 1) [4, 5, 6, 7].
Within a module, the domains work together to incorporate the incoming amino acid into the growing peptide (Figure 1b) [5, 8]. A basic elongation module contains a condensation (C) domain, an adenylation (A) domain, and a peptidyl carrier protein (PCP) domain. The A domain selects and adenylates the cognate amino acid, then attaches it by a thioester link to a prosthetic phosphopantetheinyl (PPE) group on the PCP domain. The PCP domain then transports the amino acid to the C domain, which catalyzes amide bond formation between this amino acid and the peptide attached to the PCP domain of the preceding module, elongating the peptide by a single residue. Next, the PCP domain brings the elongated peptide to the downstream module, where it is passed off and further elongated in the next condensation reaction. Once a PCP domain has donated its peptide, it can accept a new amino acid from the A domain and participate in the next cycle of assembly-line synthesis. Initiation modules lack the C domain, and termination modules usually contain a thioesterase (Te) domain, which releases the peptide by cyclization or hydrolysis. A canonical organization of a basic NRPS is A–PCP-(C–A–PCP)n–Te (Figure 1c). Additionally, NRPS modules very often have tailoring domains, including oxidase, reductase, epimerization, ketoreductase, aminotransferase and methyltransferase domains, and the action of these domains is incorporated into the catalytic cycle of the module [9]. NPRSs can alternatively end in a reductase [10•] or terminal C domain [11•, 12]. This wide range of tailoring domains, combined with the over five hundred monomers that can be used as substrates, including D-amino acids, aryl acids, hydroxy acids, and fatty acids, allows nonribosomal peptides to occupy a diverse area of chemical space [13].
Starting with Conti's determination of an A domain in 1997 [14], each core NRPS domain has been structurally characterized [5, 15]. The A domain has a large Acore (∼450 amino acids) portion with binding sites for ATP and substrate amino acid, and a small Asub (∼100 amino acids) portion that changes position depending on functional state [14, 16, 17, 18, 19, 20]. (Acore and Asub are also called the large/N-terminal and small/C-terminal subdomains [21].) The C domain is a ∼450 amino acid V-shaped pseudodimer of chloramphenicol acetyltransferase folds, with an active site at the middle of a tunnel connecting binding sites for donor and acceptor PCP domains. The PCP domain is an oblong 4-helix bundle of ∼80 amino acids, homologous to fatty acid synthase and polyketide synthase acyl carrier protein domains, with the PPE attached to a conserved serine at one end. The Te domain is a ∼275 amino acid α/β hydrolase domain with an active site topped by a variable ‘lid’ region. Studies investigating the catalytic mechanisms of each individual reaction in the NRPS cycle are reviewed elsewhere [5, 15, 16, 22, 23]. Furthermore, some tailoring domains have been structurally characterized, several didomain structures have been determined, and a landmark study in 2008 from Marahiel's lab produced a snapshot of an entire termination module (reviewed in [5]). However, a most intriguing aspect of NRPS function is how these domains transiently and productively interact during the catalytic cycle and how domains are built up into modules and full NRPS megaenzymes [8]. Multiple structures of large constructs of NRPSs in recognizable functional states are required to understand these megaenzymes. Over the last ∼2 years, several studies, including on full modules and module-sized constructs of NRPSs, have started to reveal the catalytic cycle in the context of the large macromolecular machine. In this review, we follow a hypothetical nascent peptide from initiation to termination, with special emphasis on insight gained from these recently-determined structures of large NRPS proteins.
Section snippets
Start it off
Minimal initiation modules contain only A and PCP domains. However, it is not uncommon for NRPSs to start with more complex initiation modules. Lipopeptides synthetases for peptide like daptomycin contain starter C domains that use acyl-PCPs as donor substrates; depsipeptide synthetases for valinomycin and cereulide contain ketoreductase domains; and kolossin A (Figure 1) and linear gramicidin synthetase contain formylation (F) domains. Recently, we determined a series of crystal structures of
Pass it on
The (formyl-)aminoacyl-PCP moves from the initiation module to the donor site of the C domain. This donor site was identified by apo structures of the C domain and biochemically [31, 32]. The structure of an excised PCP–C didomain from TycC visualized the two domains required for donation, but was in a non-productive conformation [33]. However, recently two didomain structures of PCP and C domain homologues, a terminal cyclizing C (CT) [11•] and an epimerization (E) domain [34•] produced the
Keep it going
The first view of any module was that of the termination module of surfactin synthetase, SrfA-C, solved in the peptide-accepting state almost a decade ago [35]. With a domain architecture of C–A–PCP–Te, it represents both a minimal C–A–PCP elongation domain and the most common type of bacterial termination module. The SrfA-C structure showed very large distances between active sites in NRPSs indicating that substantial conformations changes would occur in the catalytic cycle (Figure 3a-i).
Finish him!
This elongation cycle (or an expanded cycle with a tailoring step) occurs in every canonical elongation module along the NRPS assembly line, until the termination module. Here, after one more elongation cycle, the PCP domain delivers the elongated peptide to the chain-terminating domain. In fungal termination modules, that is often a CT domain, and the PCP–CT structure is as discussed above, but for bacterial NRPSs, it is most commonly a Te domain and second most commonly a terminal reductase
Bring it all together
The above-described structures, in context of excellent existing functional studies, provide a wealth of insight into each step of the NRPS cycle. They are less informative about how modules form intact NRPS megaenzymes. High resolution structures of multimodular NRPSs are an outstanding goal in the field, and multiple such structures are required to answer questions of NRPS architecture, but a view of higher order structure is beginning to form.
We recently solved the structure of a
Onward
Recent work has illuminated many aspects of NRPSs structure. The next several years will likely be equally insightful. It will be interesting to see whether the coming structures of multimodular NRPSs will be determined by crystallography or cryo-EM. Cryo-EM is now the technique of choice for large macromolecules but the moderate size of NRPS domains and especially the massive array of domain–domain and module–module conformations which are absolutely required for NRPS function (many of which
Conflict of interest statement
Nothing declared.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgments
We thank the Schmeing lab for helpful discussions. Funding to TMS from the Canadian Institutes of Health Research (FDN-148472), Canada Research Chairs, and the Natural Sciences and Engineering Research Council (Discovery Grant 418420-12) is gratefully acknowledged.
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Equal contribution.