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Biosynthesis of Natural Products Nonribosomal Peptides

Biosynthesis of Natural Products Nonribosomal Peptides. 5-19-2011. Peptide Natural Products. Highly stable multicyclic peptide antibiotics. Precursor peptide undergoes posttranslational side chain modification reaction involving amino acid modifications and removal of leader peptide.

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Biosynthesis of Natural Products Nonribosomal Peptides

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  1. Biosynthesis of Natural ProductsNonribosomal Peptides 5-19-2011

  2. Peptide Natural Products Highly stable multicyclic peptide antibiotics. Precursor peptide undergoes posttranslational side chain modification reaction involving amino acid modifications and removal of leader peptide. • Ribosomally Derived Nisin – a lantibiotic thiopeptides Walsh, JACS 2010

  3. Formation of Lantibiotics Van der donk, Nat. Chem. Biol. 2010

  4. Formation of Thiopeptides Van der donk, Nat. Chem. Biol. 2010

  5. Nonribosomal Peptides tyrocidine A vancomycin penicillins cyclosporin daptomycin

  6. Nonribosomal Peptides (NRPs) • A majority of peptidyl natural products are nonribosomally derived – they are not synthesized from the protein translational machinery. Instead they are assembled via the assembly-line like actions of nonribosomal peptide synthetases (NRPS) • NRPs can include many nonproteinogenic amino acids, including hydroxyl acid, N-methylated aa, and D-aa; whereas ribosomally derived peptides are restricted to 20 naturally occurring amino acids. • The biosynthetic logic of NRPS parallels that of modular PKSs: template-directed, colinearity, assembly-line like. • NRPS can also be coupled to PKS assembly lines (both emply thioester acyl intermediates) to give hybrid compounds.

  7. Basic Enzymatic Units of NRPS SH adenylation thioesterase Surfactin NRPS Module 7 (144 kDa) C A PCP TE condensation peptidyl carrier protein Marahiel, Science 2008

  8. Basic Biochemistry – PCP domain The PCP protein is structurally and functionally analogous to the ACP domain. The apo form of PCP must be modified at a serine side chain to become holo form. The holo form contains a phosphopantetheinyl arm and a nucleophilic thiol, which is the position at which the peptidyl groups will be attached. Sometime also referred to as the thiolation (T) domain. PPTase PCP PCP PCP Konig, Science 2006

  9. Basic Biochemistry – A domain The A (adenylation) domain selects and activates the building blocks. The building blocks are mostly amino acids, both proteinogenic and nonproteinogenic. They can also be other organic acids. Two step activation: 1) adenylation with ATP; 2) transfer to PCP domain. The adenylation step is identical to that of aminoacyl-tRNA synthase. 1) Adenylation A SH 2) Acyltransfer PCP PCP A Aminoacyl-S-PCP

  10. A domain specificity Representative nonproteinogenic amino acids selected by the NRPS A domain

  11. A-Domain Specificity Stachelhaus, Chem Biol, 1999

  12. Basic Biochemistry – C domain Catalyzes formation of peptide bonds between growing NRP and aminoacyl-S-PCP. Upstream peptidyl-S-PCP is the acyl donor. Downstream, intramodular aminoacyl-S-PCP as the nucleophile acceptor. Peptide-bond formation is unidirectional. Functionally analogous to the KS domain in PKSs and makes C-N bonds. PCPn C A PCPn+1 PCPn C A PCPn+1 Samel, Structure 2007

  13. Accessory Domains – Cyclization (Cy) and Oxidation (Ox) Cy domains are responsible for the formation of heterocycles in NRPs. Cy domains are variants of C domains and also catalyze peptide bond formation. Cy domains then catalyze the attack of b-nucleophile on the upstream amide carbonyl, and form five membered rings that dehydrates. b-nucleophile: serine (hydroxyl)  oxazoline; cysteine (thiol)  thiazoline. Further oxidation by the Ox domain can lead to oxazole or thiazoles PCP Cy A PCP PCP Cy A PCP PCP Cy A PCP condensation cyclization dehydration PCP Cy A PCP PCP Cy A Ox PCP epothilone Oxidation FAD dependent

  14. Accessory Domain – Epimerization (E) D-amino acids occur commonly in NRPs. Most of the D-amino acids arise from the action of the epimerase (E) domain. The L-amino acid is first selected and tethered to the PCP domain. The aminoacyl-S-PCP is then subjected to epimerization via extraction of the Ca-proton. The epimerization passes through the planar, resonance-stablized Ca carboanion. The downsteam C domain has stereoselectivity in choosing the epimerized substrate. A PCP E A PCP E epimerization PCP PCP

  15. Accessory Domain – N-methylation (MT) N-methyltransferase domains are embedded between the A and the PCP domains. The substrate for methylation is S-adenosylmethionine (SAM), which provides an electrophilic CH3+ equivalent. The product of the reaction is N-Me-aminoacyl-S-PCP, before the next condensation. Cy A MT PCP Cy A MT PCP Cy A MT PCP Pyochelin PchF SAM 3X C A PCP C A MT PCP PCP TE enniatin Patel, Biochemistry, 2003

  16. Basic Biochemistry – TE domain TE is the chain terminating domain, and is usually attached to the last PCP domain. It has the same role as the TE in the PKSs and serves to disconnet the covalent thioester linkage between the full length peptidyl chain and the most downstream PCP domain. TE can function both as a hydrolytic enzyme and as a macrocyclization enzyme. C A PCP TE C A PCP TE a b c Linear peptide Macrolactam Macrolactam/Macrolactone Adapted from Walsh, Antibiotics

  17. Putting it all together – NRPS modules Initiation Elongation Termination A PCP C/Cy A Ox MT PCP E C A PCP TE optional

  18. Konig, NPR 2008

  19. The principle of NRPSs is elucidated using the synthetic machinery of the branched cyclic decapeptide bacitracin A. A total of twelve modules distributed over three NRPSs (BacA, BacB and BacC) process the growing peptide chain along the protein template. During synthesis, the elongation intermediates remain covalently tethered as thioesters to the cofactors of the NRPSs. After the linear peptide has reached the final module it is released by macrocyclization. Schwarzer, NPR 2003

  20. Example – ACV Synthetase A single polypeptide NRPS from Acremonium chrysogenum that synthesizes the acyclic precursor of the penicillin and cephalosporins. ACV synthetase has 10 domains in a 450-kDa protein. Uses three building blocks (d-amino adipic acid, cysteine and valine, ACV). L-valine, Epimerized to D form d-amino adipic acid (C6-COOH is activated) L-cysteine hydrolysis AAad PCP C ACys PCP C AVal PCP E TE Penicillins Cephalosporins IPN Synthetase ACV Isopenicillin N DAOC Synthetase Deacetoxycephalosporin C (DAOC) Both IPN and DAOC synthetases are nonheme ferrous iron dioxygenases

  21. Example - Vancomycin Satterly, NPR, 2008

  22. Post NRPS modification Oxidative Coupling Glycosylation

  23. PKS-NRPS hybrids Many natural products are hybrids of nonribosomal peptides and polyketides. Colinearity rule still applies to these mosaic assembly line. All intermediates are still shuttled via pPant arms of ACP or PCP domains. Epothilone biosynthetic gene cluster Tang and Khosla, Science 2000

  24. Case Study - Daptomycin Asp9 Gly10 D-Ala8 D-Ser11 Asp7 DptBC Orn6 MeGlu12 Isolated from Streptomyces roseosporus in 1987, Daptomycin is an acidic cyclic lipopeptide antibiotic approved for treatment of infections caused by Gram-positive pathogens, including Staphylococcus aureus strains resistant to other antibiotics. Gly5 Kyn13 Thr4 DptD Asp3 Structurally, daptomycin is composed of two portions: NRP core and a tethered fatty acyl chain. The NRP core contains thirteen amino acids, including three D-amino acids (D-Asn2, D-Ala8, D-Ser11)and three non-proteinogenic amino acid (ornithine6, (2S, 3R)-3-methylglutamic acid12, kynurenine13). The decanoyl moiety is linked to the N-terminus of the first residue tryptophan. D-Asn2 R Trp1 DptA

  25. Mode of action MRSA MSSA Staphylococcus aureus is a facultatively anaerobic, gram positive coccus and is the most common cause of staph infections. Methicillin-sensitive Staphylococcus aureus (MSSA) refers to all of the antibiotic(beta-lactam)-sensitive strains of Staph aureus. Staphylococcus aureus Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium responsible for several difficult-to-treat infections in humans. MRSA is, by definition, any strain of Staphylococcus aureus bacteria that has developed resistance to beta-lactam antibiotics, which include the penicillins (methicillin, dicloxacillin, nafcillin, oxacillin, etc.) and the cephalosporins In a first step, daptomycin aggregates in solution in the presence of a minimum of 1:1 calcium to daptomycin molar ratio. Then, daptomycin inserts into the membrane, a process facilitated by calcium, which binds strongly to phosphatidylglycerol headgroups. At this point, daptomycin induces membrane depolarization without cell lysis. As a result, loss of membrane potential leads to inhibition of protein, DNA, RNA synthese and finally death of cells. Straus and Hancock, BBA

  26. dptgene cluster and NRPS organization Truncated depiction of the daptomycin assembly line illustrating modules 1 and 13. Nolan and Walsh, CBC

  27. Related Lipopeptides Baltz, Methods Enzymol and Miao, Chem. Biol.

  28. Combinatorial Biosynthesis Baltz, Curr. Opin. Chem. Biol.

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