Nonribosomal peptide synthetases involved in the production of medically relevant natural products

Abstract

Natural products biosynthesized wholly or in part by nonribosomal peptide synthetases (NRPSs) are some of the most important drugs currently used clinically for the treatment of a variety of diseases. Since the initial research into NRPSs in the early 1960s, we have gained considerable insights into the mechanism by which these enzymes assemble these natural products. This review will present a brief history of how the basic mechanistic steps of NRPSs were initially deciphered and how this information has led us to understand how nature modified these systems to generate the enormous structural diversity seen in nonribosomal peptides. This review will also briefly discuss how drug development and discovery are being influenced by what we have learned from nature about nonribosomal peptide biosynthesis.

Figure 1

(A) Schematic of the historical “thiotemplate” mechanism for NRPSs. The E1 represents the 4'-Ppant-containing portion of the enzyme, with the 4'-Ppant represented by the squiggled line. E2 and E3 represent modules of the NRPS that contain a corresponding aminoacylthioester. E1 is used repeatedly through the biosynthesis of the nonribosomal peptide, while the individual modules such as E2 and E3 are used once. (B) Schematic of the “multiple carrier model” mechanism for NRPSs. E1, E2, and E3 represent individual modules containing individual aminoacylthioesters that are condensed in a directional manner to generate the nonribosomal peptide product.

Figure 2

Schematic representations of the reactions catalyzed by each of the core domains of NRPSs. The domain involved in the reaction shown at the right is highlighted in black on the left. (A) adenylation domain, catalyzing aminoacyl-AMP formation; (B) thiolation domain, highlighting the formation of the aminoacylthioester; (C) condensation domain, showing the formation of a peptide bond between two aminoacylthioester substrates. The T1 and T2 denote the T domains from neighboring NRPS modules. The “d” and “a” shown within the black C domain denote the donor and acceptor sites, respectively; (D) thioester domain, catalyzing first aminoacylester formation on the Te domain followed by either hydrolysis or cyclization of the peptide. The “X” represents either a nitrogen or oxygen. The 4'-Ppant cofactor is represented by the SH bonded to each T domain. From this figure forward, 4'-Ppant cofactor will be represented in this manner.

Figure 3

Schematic representation of the ten module tyrocidine NRPS. The ten modules are distributed over three peptides. Throughout this review, each module is numbered and underlined by a bar to denote the domains associated with each module. The amino acid linked directly to the T domain is the amino acid activated by the associated module. The standard three letter abbreviation for each standard amino acid is used, with L-Orn representing L-ornithine. The peptides tethered to each T domain represent the growing peptide tethered to each T domain.

Figure 4

Schematic representation of the NRPS, ACV synthetase, that assembles the ACV tripeptide intermediate. The amino acid abbreviation below each module identifies the amino acid activated by that module (L-Aad, L-α-aminoadipate). Isopenicillin N is a common intermediate for the formation of penicillin G and cephalosporin C.

Figure 5

Schematic representation of the daptomycin NRPS. The initiating module (IM) consists of DptE (AL, acyl-CoA ligase homolog) and DptF (T, ACP T domain). The remaining NRPS components are contained on DptA, DptBC, and DptD. Abbreviations of nonproteinogenic substrates: Dec, decanoic acid; L-Orn, L-Ornithine; 3mGlu, 3-methyl-L-Glu; Kyn, kynurenine.

Figure 6

Structures of daptomycin, the related lipopeptides A54145 and calcium-dependent antibiotic, and daptomycin analogs generated through combinatorial biosynthesis. For the analogs, the residues that have been found at each position are noted, including natural variations in the lipid. Abbreviations for unusual amino acids: L-Orn, L-ornithine; 3mGlu, 3-methyl-L-Glu; mOAsp, 3-O-methyl-Asp; hasp, 3-hydroxy-L-Asp; Sar, sarcosine; Kyn, kynurenine.

Figure 7

Schematic representation of the cyclosporin A NRPS. Abbreviations for nonproteinogenic amino acids: Bmt, (4R)-4-[(E)-2-butyl]-4-methyl-L-Thr; Aba, 2-amino-L-butyric acid

Figure 8

Schematic representation of the chloroeremomycin NRPS and the timing of the oxidative crosslinking of the heptapeptide backbone. The oxidative crosslinking between residues 2-4, 4-6, and 5-7 are noted. The crosslinking is shown occurring while the heptapeptide is thioesterified to the T domain of module seven. The “X” on the L-Bht residues are either H or Cl depending on the timing of chlorination. Abbreviations of nonproteinogenic amino acids: L-Bht, β-hydroxy-L-Tyr; L-Hpg, L-p-hydroxyphenylglycine; L-Dhp, 3,5-dihydroxy-L-phenylglycine. The structures of vancomycin and teichoplanin are shown at the bottom right. For these glycopeptides the NRPSs and downstream modifying enzymes will differ with those seen in chloroeremomycin to reflect the structural differences.

Figure 9

Schematic representation of the echinomycin NRPS. The dimerization of each monomer is shown occurring on module five of the NRPS. The initiating module consists of the proteins Ecm1 and FabC, noted as A and T domains of module one, respectively, while the remaining components are on Ecm6 and Ecm7. Abbreviation: Qoa, quinoxaline.

Figure 10

Schematic representation of the capreomycin NRPS and the formation of the four components of capreomycin. The NRPS consists of CmnF, CmnA, CmnI, and CmnG, along with the acyl-CoA dehydrogenase homolog CmnJ (denoted by DH). The NRPS domains are abbreviated as in the text with the addition of “X” representing a domain potentially involved in L-Ser-to-L-Ala conversion (or module two activates either amino acids; R = H or OH), and “C/” that represents a modified C domain involved in cyclization of the pentapeptide. The arrows below the NRPS connecting the A domain of module one with the T domains of modules one and four denote the nonlinearity of the NRPS. The monomodular NRPS involved in β-Lys addition is shown on the right, with CT representing the carbamoyltransferase CmnL.

Figure 11

Schematic representation of the bleomycin NPRS/PKS megasynthase. The PKS portion of the megasynthase consists of module 7. Abbreviations not in text: KS, ketosynthase; AT, acyltransferase, MT; methyltranferase; KR, ketoreductase; Ox, oxidase; C’, potentially inactive C domain; Mal-CoA, malonyl-CoA; β-Ala, β-alanine; AL, acyl-CoA ligase. The “?” indicates the unknown function of the first module.