5.451 F2005 Natural Product Biosynthesis Relevance of natural products in the pharmaceutical industry (see Table 2 in Butler J. Nat. Prod. 2004, 67, 2141-2153.)) Table removed due to copyright reasons. 2* a phosphopantetheinyl transferase attaches a phosp. group of CoA to serine in PCP (or T) * provides a long (20 Å) thiolate handleA domain kind of like a codon in Ribosomal system 5.451 F2005 The specificity of the amino acid is encoded into the A domain protein sequencePeptide BiosynthesisTable 1 in Chem. Biol. (1999) 6, 493-505.Non-Ribosomal Peptides Chem. Biol. (2000) 7, 211-224. Redesign efforts:J. Comp. Biol.2005, 12, 740-761. 1) "swap" A domains from another pathway --> interferes w/ protein iinteractions found in remainder of pathway 2) point mutations. (SDM). 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 Table removed due to copyright reasons. 65.451 F2005 Peptide BiosynthesisNon-Ribosomal Peptide Biosynthesis: 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 cyclization- may 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)Oxidation of thiazoline/oxazoline to thiazole/oxazole (Ox domain)Reduction of thiazoline/oxazoline to thiazolidone/oxazolidone (Red domain)N-methylation of amide amine(MT domain)Epimerization of L amino acid to D amino acid(E domain)Hydroxylation at the beta carbon position of the amino acid side chain (Fe containing Ox domain) The protein domains that catalyze these reactions are typically adjacentto 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 105.451 F2005 Peptide Biosynthesis Non-Ribosomal Peptides Small molecules that are attached to proteins that facilitate particularly difficult chemistry Cofactors to recognize and the reactions they catalyze: Oxidation: Reduction: HHO HO flavin NADPH NH2NH2 R RH ONN NN O N ON O R NH NH OH OH N N NH2O HO OPO ON N OPO N NO O OH OPO32-Oxidation: Iron, with the appropriate ligands Methylation:H SAMNO His OH2 NH2 NH2N O Asp N NNFe NH2O OH2 N NN NNS SO CO2 CO2 HN His H2N O H2N OH OH Heme Non-Heme CO2H CO2H OH OH 13 N N NN Fe 3+5.451 F2005 Peptide BiosynthesisNon-Ribosomal Peptides Small molecules that are attached to proteins that facilitate particularly difficult chemistry Cofactors to recognize and the reactions they catalyze: (not used in NRPS but see in other 2˚ metabolic pathways- will encounter later) Transamination: pyridoxal phosphate CO2H R NH2 B: HR CO2 BH+ R CO2 NH NH H O O OO O3PO O3PO O3PO N HH H NN Carbanion for decarboxylations: thiamine-PP (TPP) NH2 N N N SCO2-NH2 N N decarboxylations, etc. N S OP2O63-OP2O63-H B: O 14mid 1950s isolated enzyme from human lines --> added CO, UV absorbance shifted to 450 --> hence P450 5.451 F2005 Peptide BiosynthesisNon-Ribosomal PeptidesElaborating the structures produced by the peptide synthetasesCatalytic Domains added into the primary amino acid sequence Hydroxylation by P450 enzymes OH OO S H2NOC NH2NADPH NADP+ R H OR S N e-NH2 CONH2NH2 Fd Fd Red NH e-SN NO NOH HN NA PCP A PCP Fe-Ox NH2N NH N S+Me2HMe O N OH OHO SO2 H2O OH O HOH2C OO NH BleomycinOH NN OH O OH Fe OCONH2 N OH N Hydroxylation at beta carbon 1) handle (bleo) 2) further rearrangement --> novobiocin CO2 CO2 Hijacking of the NRPS/hydroxylation machinery to make non-peptide structuresUsing a thioester tethered system to make a dedicated pool of non-proteogenic amino acids O HH2N O OH NH OH OH HO N OOH NH2 NHOO NO OOO O O OH N Me OHO 17OH OH Novobiocin Nikkomycin5.451 F2005 Peptide BiosynthesisNon-Ribosomal PeptidesElaborating the structures produced by the peptide synthetasesCatalytic Domains added into the primary amino acid sequence Hydroxylation by P450 enzymes: Basic idea of how heme works Figure removed due to copyright reasons. Scheme 4.27. Silverman, The Organic Chemistry of Enzyme Catalyzed Reactions. 18Chlorination most common Bromination in marine based Nat. Products Limited fluorinases 5.451 F2005 Peptide BiosynthesisNon-Ribosomal PeptidesCatalytic Domains added into the primary amino acid sequence Halogenation (Chlorination)PNAS, 2005, 102, 10111-10116 Fluorinase Nature (2002) 416, 279 Copyright 2005 National Academy of Sciences, U.S.A. 205.451 F2005 Peptide BiosynthesisNon-Ribosomal PeptidesCatalytic Domains added into the primary amino acid sequence Halogenation (Chlorination)PNAS, 2005, 102, 10111-10116 Cl OO HO A PCP NH2 SHO A PCP NH2 S NRPS synthesisSyrB2L-Threonine SyrB1 halogenase iron containing protein non heme iron 21 Copyright 2005 National Academy of Sciences, U.S.A.22 5.451 F2005 Peptide BiosynthesisNon-Ribosomal PeptidesCatalytic Domains added into the primary amino acid sequence Halogenation (Chlorination)PNAS, 2005, 102, 10111-10116 cofactor alpha keto glutarate generate radical using activated oxygen --> CO2 oxygenase --> hydroylates oxidadase --> oxidation (not necessarily hydroxylation) activated oxygen species Copyright 2005 National Academy of Sciences, U.S.A.5.451 F2005 Peptide Biosynthesis Non-Ribosomal Peptides Elaborating the structures produced by the peptide synthetases HH H2N N H2NNS Sβ-lactam N CO2-O O NCOOH O O CO2H CO2-Isopenicillin N Deacetoxycephalosporin C HS OCOOH O O HO H2N H2N N SH2N NH2N S SS HCOOH O OSH A PCP C A PCP C A PCP E TE PCP adiapic acid cysteine valine TE O H SFe E O H2NN H OH OH2NN HN HCOOH O N H2N N OH NO COOH O O HHO2C S Oelectrophile Fe O INS COOH O Ring 1 SH E Nucleophile OH H H2N NS Nature (1998) 394, 805.H H2N N SFe E Nature (1999) 401, 721.COOH O N COOH O NO O Isopenicillin N CO2H 23 HO2C5.451 F2005 Peptide BiosynthesisNon-Ribosomal PeptidesElaborating the