Table removed due to copyright reasons.

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2 atural Product Biosynthesis Relevance of natural products in the pharmaceutical industry (see Table 2 in Butler J. at. Prod. 2004, 67, )) Table removed due to copyright reasons. 2

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4 * a phosphopantetheinyl transferase attaches a phosp. group of CoA to serine in PCP (or T) * provides a long (20 Å) thiolate handle

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6 A domain kind of like a codon in Ribosomal system F2005 on-ribosomal Peptides The specificity of the amino acid is encoded into the A domain protein sequence Table 1 in Chem. Biol. (1999) 6, Chem. Biol. (2000) 7, Redesign efforts: J. Comp. Biol. 2005, 12, ) "swap" A domains from another pathway --> interferes w/ protein iinteractions found in remainder of pathway 2) point mutations. (DM). Table removed due to copyright reasons. Phe --> Leu Asp --> Asn Glu --> Gln *Phe A domain crystallized --> contacts w/substrate noted 10 protein aa's that made contect *can predict which aa activated by substrate 6

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10 on-ribosomal : Can a biosynthetic pathway be predicted from the structure??? 1. Recognize the amino acid building blocks and trace out the amide-bond backbone chain. 2. The peptide natural product is most likely made by the nonribosomal peptide synthetases if: Its cyclic. Look for the point of cyclizationmay be an ester. may be an amide bond formed with an amine side chain (I.e. Lys) Its a small peptide (less than 20 residues) It has unusual amino acids (A domains can incorporate unusual amino acids) or D amino acids 3. Identify the modifications to the basic peptide structure. The most common modifications are: Cyclization of cysteine, serine, threonine to thiazoline, oxazoline (Cy domain that replaces C domain) xidation of thiazoline/oxazoline to thiazole/oxazole (x domain) Reduction of thiazoline/oxazoline to thiazolidone/oxazolidone (Red domain) -methylation of amide amine(mt domain) Epimerization of L amino acid to D amino acid(e domain) ydroxylation at the beta carbon position of the amino acid side chain (Fe containing x domain) The protein domains that catalyze these reactions are typically adjacent to the C-A-PCP core domains that make the amide bond Addition of lipids, fatty acids, sugars happen after the core of the peptide has been formed, and the peptide has been released from the peptide synthetase by the thioesterase (TE) domain. We will discuss glycosylation later 10

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13 on-ribosomal Peptides mall molecules that are attached to proteins that facilitate particularly difficult chemistry Cofactors to recognize and the reactions they catalyze: xidation: Reduction: flavin ADP 2 2 R R R P P 2 P 3 2- xidation: Iron, with the appropriate ligands Methylation: AM 3+ is Fe Asp Fe 2 2 C 2 C is eme on-eme C 2 C 2 13

14 on-ribosomal Peptides mall molecules that are attached to proteins that facilitate particularly difficult chemistry Cofactors to recognize and the reactions they catalyze: (not used in RP but see in other 2 metabolic pathways- will encounter later) Transamination: pyridoxal phosphate C 2 R R 2 B: C2 B + R C 2 3 P 3 P 3 P Carbanion for decarboxylations: thiamine-pp (TPP) 2 2 B: P C 2 - P decarboxylations, etc. 14

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17 mid 1950s isolated enzyme from human lines --> added C, UV absorbance shifted to > hence P F2005 on-ribosomal Peptides Elaborating the structures produced by the peptide synthetases Catalytic Domains added into the primary amino acid sequence ydroxylation by P450 enzymes ADP ADP + R 2 C 2 R e Fd C2 Fd Red e - A PCP Fe-x A PCP 2 Me C Bleomycin Fe C 2 C 2 C 2 ydroxylation at beta carbon 1) handle (bleo) 2) further rearrangement --> novobiocin + Me 2 ijacking of the RP/hydroxylation machinery to make non-peptide structures Using a thioester tethered system to make a dedicated pool of non-proteogenic amino acids 2 ovobiocin 2 Me ikkomycin 17

18 on-ribosomal Peptides Elaborating the structures produced by the peptide synthetases Catalytic Domains added into the primary amino acid sequence ydroxylation by P450 enzymes: Basic idea of how heme works Figure removed due to copyright reasons. cheme ilverman, The rganic Chemistry of Enzyme Catalyzed Reactions. 18

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20 Chlorination most common Bromination in marine based at. Products Limited fluorinases F2005 on-ribosomal Peptides Catalytic Domains added into the primary amino acid sequence alogenation (Chlorination) PA, 2005, 102, Fluorinase ature (2002) 416, 279 Copyright 2005 ational Academy of ciences, U..A.

21 on-ribosomal Peptides Catalytic Domains added into the primary amino acid sequence alogenation (Chlorination) PA, 2005, 102, Cl 2 2 L-Threonine A PCP yrb1 yrb2 A PCP halogenase iron containing protein non heme iron RP synthesis 21 Copyright 2005 ational Academy of ciences, U..A.

22 on-ribosomal Peptides Catalytic Domains added into the primary amino acid sequence alogenation (Chlorination) PA, 2005, 102, cofactor alpha keto glutarate generate radical using activated oxygen --> C 2 oxygenase --> hydroylates oxidadase --> oxidation (not necessarily hydroxylation) activated oxygen species 22 Copyright 2005 ational Academy of ciences, U..A.

23 on-ribosomal Peptides Elaborating the structures produced by the peptide synthetases β-lactam 2 2 C - 2 C C 2 C - 2 Isopenicillin Deacetoxycephalosporin C C C A PCP C A PCP C A PCP E TE PCP adiapic acid cysteine valine TE 2 Fe E 2 C 2 C 2 C I C electrophile Fe Ring 1 E ucleophile 2 Fe E C C Isopenicillin 2 C 2 ature (1998) 394, 805. ature (1999) 401, 721. C 2 23

24 on-ribosomal Peptides Elaborating the structures produced by the peptide synthetases 2 C C 2 Deacetoxycephalosporin C 2 epimerization 2 DAC 2 C C C non heme iron C 2 C 2 Isopenicillin substrate α KG cofactor C 2 Ring expansion 2 2 C C C 2 C 2 24

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26 on-ribosomal Peptides Elaborating the structures produced by the peptide synthetases P450 enzymes also catalyze C- and C-C bond formation: xidative Crosslinking Figure removed due to copyright reasons. ee cheme 24 in Angew. Chem. Intnl. Ed. (2003) 42,

27 on-ribosomal Peptides Elaborating the structures produced by the peptide synthetases Cl Cl Cl Cl CepF C 3 C CepE Cl Cl Cl Cl CepG C 3 C ever reconstituted in vitro Timing unclear --> scale up revealed lots of truncated peptide products Genetic knockouts --> analyze culture broth for products 27

28 on-ribosomal Peptides Review of Peptide Modifications 1. Cyclization (replaces C domain): amide bond formation (not shown) and then cyclization/dehydration Cys to thiazoline er to oxazoline Thr to methyloxazoline example: yersiniabactin 2. xidation thiazoline to thiazole (oxazoline to oxazole) note: uses flavin as oxidizing agent examples: bleomycin, epothilone 3. Reduction thiazoline to thiazolidine (oxazoline to oxazolidine) note: uses ADP as reducing agent example: yersiniabactin 28

29 on-ribosomal Peptides Review of Peptide Modifications 4. -Methylation (MT) 2 A MT PCP A MT PCP note: uses AM as methyl source example: cyclosporin 5. Epimerization A PCP E A PCP E A PCP E example: gramicidin 6. ydroxylation 2 x-fe A PCP 2 x-fe A PCP note: uses heme-chelated iron as oxidant example:bleomycin, ramoplanin, novobiocin 29

30 on-ribosomal Peptides Review of Peptide Modifications 7. alogenation (chlorination most common) 2 Cl 2 note: non-heme-chelated iron as oxidant example:syringomycin x-fe A PCP x-fe A PCP Major changes to the peptide backbone structure are typically carried out after peptide biosynthesis Most important examples: xidative crosslinking in vancomycin biosynthesis b-lactam formation in penicillin/cephlasporin biosynthesis Also occuring after peptide biosynthesis: Glycosylation of peptide (will be discussed in detail later) Derivatization with lipids 30

31 Ribosomal Peptides standard ribsosome-based protein synthesis of standard linear peptide proteolysis and post-translational modification occurs after protein is translated examples are microcin and lantibiotics (most peptide based natural products are synthesized non-ribosomally ) Lantibiotics Chem. Rev. 2005, 105,

32 Ribosomal Peptides Lantibiotics standard peptide biosynthesis on ribosome Figure removed due to copyright reasons. Please see Figure 4 in Chem Rev 105 (2005):

33 Ribosomal Peptides Lantibiotics Figures removed due to copyright reasons. Please see Figures 22 and 23 in Chem Rev 105 (2005):

34 on-ribosomal Peptides What is a gene cluster? The genes that encode the biosynthetic enzymes next to each other on a chromosome Gene = open reading frame (RF) = ATG XXX XXX XXX TAA start/stop codon in frame (multiple of 3) Figure removed due to copyright reasons. prokaryotes (i.e. bacteria) many yeast/fungi (simple eukaryotes) Figure 3 in Microbiology, 2005, 151, Daphomycin Biosynthesis 34