5. Polyketides RA Macahig FM Dayrit

Introduction Polyketides (which literally means “many ketone groups”) make up a diverse biogenetic group which starts from acetyl-CoA to form a linear chain without extensive reduction. The polyketide chain can cyclize to form aromatic rings or undergo extensive derivatization. Polyketides rank among the largest group of secondary metabolites in terms of diversity of structure and biological diversity. Polyketide biosynthesis shares some similarities with the initial steps of fatty acid acetyl polymerization. Like the fats, the polyketide pathway probably arose early in biological evolution before the rise of plants. 5. Polyketides (Dayrit)

Examples of polyketide natural products which illustrate the wide variety of structures which comprise this group. 5. Polyketides (Dayrit)

Introduction The polyketides have great diversity of structures and chemical functionalities. These structures range from saturated macrocyclic lactones (macrolides), which are unique polyketide metabolites, to various types of aromatic compounds. Polyketides occur widely in bacteria, fungi and lichens, but are of relatively minor occurrence in higher plants. Bacteria, in particular Actinomycetes and Cyanobacteria, are prolific sources of polyketides, many of which possess antibiotic activity. Other significant polyketide producers are Aspergillus (aflatoxins) and Penicillium and Streptomyces species (tetracycline antibiotics). 5. Polyketides (Dayrit)

Overview of polyketide biosynthesis Polyketides are produced from poly-acetyl intermediates (poly-1,3-diketo compounds) which do not undergo complete reduction, as in the case of the fats. The polyketides then branch into two major pathways: Aromatic compounds. The reactive 1,3-diketo groups undergo intramolecular Claisen or lactonization reactions forming cyclic compounds. Dehydration produces aromatic compounds. Macrolides. The keto- groups are reduced to alcohols, which are subsequently dehydrated to form linear compounds. The final products are macrocyclic esters. Macrolides generally >12 carbon atoms in the ring. 5. Polyketides (Dayrit)

Aromatic polyketides. Major cyclization pathways for a tetraketide followed by aromatization. 5. Polyketides (Dayrit)

Biosynthetic studies on polyketides (Arthur Birch) The elucidation of the polyketide pathway was pioneered by Arthur Birch in 1953. Birch used 14C and 18O-labeled acetate which he fed to microorganisms to establish the incorporation pattern and from this to postulate the steps in the biosynthesis of polyketides. 5. Polyketides (Dayrit)

Overview of polyketide biosynthesis Birch proposal for polyketide biosynthesis: 1. Starting with a starter unit, C2 units are added to form the polyketide chain (chain assembly). 2. Reduction and/or alkylation of the polyketide chain before cyclization. 3. Intra- or intermolecular cyclization. (The more common pathway is intramolecular cyclization.) 4. Secondary processes which modify the intermediate product after cyclization, such as: halogenation, O- methylation, C-methylation, reduction, oxidation, decarboxylation and skeletal rearrangement. 5. Polyketides (Dayrit)

Variations in number of C2 units and mode of cyclization Short-hand representation of polyketides: Variations in number of C2 units and mode of cyclization 5. Polyketides (Dayrit)

Variations in number of C2 units and mode of cyclization 5. Polyketides (Dayrit)

Variations in number of C2 units and mode of cyclization 5. Polyketides (Dayrit)

Variations in number of C2 units and mode of cyclization 5. Polyketides (Dayrit)

Inter- vs. intramolecular cyclization: A. Colletodiol; B. Use of labeling experiments to distinguish intra- from intermolecular cyclization. 5. Polyketides (Dayrit)

(from: The World of Polyketides, http://linux1.nii.res.in/) Biosynthesis of macrolides: Step-wise chemical transformations and enzymes. 5. Polyketides (Dayrit)

Hypothetical scheme of the biosynthesis of phenol polyketides on the Polyketide Synthase (PKS) multienzyme complex. 5. Polyketides (Dayrit)

Polyketide synthase (PKS) The PKS family share a number of characteristics with the family of fatty acid synthases (FAS): the PKS is a multienzyme complex which is arranged so that the stepwise transformations are carried out sequentially. ACP: acyl carrying protein KS: b-keto acyl synthase MAT: malonyl (acyl) transferase DH: dehydratase ER: enoyl reductase KR: keto reductase TE: thiol esterase (from: The World of Polyketides, http://linux1.nii.res.in/) Hypothetical model for one type of PKS multienzyme system which produces 6-methylsalicylic acid and lovastatin. The growing chain is assembled on two multienzyme complexes. 5. Polyketides (Dayrit)

The biosynthetic pathway for the fungal polyketide 6-methylsalicylic acid (6-MSA). 6-MSA is assembled from four ketide units (one acetate and three malonates). 6-MSAS contains the following domains (in order): KS, MAT, DH, KR and ACP. These act repeatedly to catalyse three rounds of chain extension, carrying out different levels of reductive processing at each stage. The first condensation is followed by reaction with a second equivalent of malonate extender unit, while the second condensation is followed by reduction and dehydration of the newly-formed keto group. After the third cycle, the chain undergoes cyclisation, dehydration and enolisation. The absence of a thioesterase domain suggests that release of the chain from the PKS does not occur by hydrolysis but by an alternative mechanism which is still not verified. (Staunton and Weismann, Nat. Prod. Rep., 2001, 18, 380–416) KS: ketosynthase MAT: malonyl-acetyl transferase DH: dehydratase KR: ketoreductase ACP: acyl carrier protein

Biosynthesis of macrolides on a modular Polyketide Synthase (PKS) multienzyme complex. (from: The World of Polyketides, http://linux1.nii.res.in/) 5. Polyketides (Dayrit)

Domain organization of the erythromycin polyketide synthase Domain organization of the erythromycin polyketide synthase. Putative domains are represented as circles. Each module incorporates the essential KS, AT and ACP domains, while all but one include optional reductive activities (KR, DH, ER). The one-to-one correspondence between domains and biosynthetic transformations explains how programming is achieved in this modular PKS. (Staunton and Weismann, Nat. Prod. Rep., 2001, 18, 380–416) 5. Polyketides (Dayrit)

KS: ketosynthase AT: acyltransferase DH: dehydratase ER: enoyl reductase KR: ketoreductase ACP: acyl carrier protein TE: thioesterase Predicted domain organization of the 6-deoxyerythronolide B synthase (DEBS) proteins. KR indicates the inactive ketoreductase domain. The ruler shows the residue number within the primary structure of the constituent proteins. The linker regions are also given in proportion. (Staunton and Weismann, Nat. Prod. Rep., 2001, 18, 380–416) 5. Polyketides (Dayrit)

KS: ketosynthase AT: acyltransferase DH: dehydratase ER: enoyl reductase KR: ketoreductase ACP: acyl carrier protein TE: thioesterase Inactivation of KR5 of DEBS results in the production of erythromycin analogues with keto groups at the C-5 position. (Staunton and Weismann, Nat. Prod. Rep., 2001, 18, 380–416) 5. Polyketides (Dayrit)

KS: ketosynthase AT: acyltransferase DH: dehydratase ER: enoyl reductase KR: ketoreductase ACP: acyl carrier protein TE: thioesterase Inactivation of ER4 results in an analogue of erythromycin with a double bond at the expected site. (Staunton and Weismann, Nat. Prod. Rep., 2001, 18, 380–416) 5. Polyketides (Dayrit)

Domain organization of the rapamycin polyketide synthase (RAPS) Domain organization of the rapamycin polyketide synthase (RAPS). As with the erythromycin PKS there is a co-linearity between the sequence of modules and the order of biosynthetic steps. (Staunton and Weismann, Nat. Prod. Rep., 2001, 18, 380–416) 5. Polyketides (Dayrit)

What is the link between FAS and PKS? The PKS system is likely derived from bacterial FAS. Different PKS pathways in bacteria illustrate the selective evolutionary advantage that multiple secondary metabolite biosyntheses confer to individual bacteria and taxonomic kingdoms. KS: ketoacyl synthase AT: acyl transferase DH: dehydratase ER: enoyl reductase ACP: acyl carrying protein Organization of fatty acid synthases (FAS) and polyketide synthases (PKS). (Jenke-Kodama et al. J Mol Bio Evol 2005) 5. Polyketides (Dayrit)

Common sequence of reactions performed by FAS and PKS. What is the link between FAS and PKS? Common sequence of reactions performed by FAS and PKS. Enzymes in a PKS module. (Jenke-Kodama et al. J Mol Bio Evol 2005) KS: ketoacyl synthase ACP: acyl carrying protein KR: ketoreductase DH: dehydratase ER: enoyl reductase 5. Polyketides (Dayrit)

Common enzymes in aromatic polyketides Four proteins comprise the minimal PKS: ketosynthase (KS), chain length factor (CLF), acyl carrier protein (ACP), and a malonyl-CoA:ACP transacylase (MAT) which is usually recruited from fatty acid synthases. Other common enzymes include: aromatase (ARO) and cyclase (CYC). (Ridley et al., PNAS, 2008, 105:4595-4600) 5. Polyketides (Dayrit)

“Deciphering the mechanism for the assembly of aromatic polyketides by a bacterial polyketide synthase,” Shen and Hutchinson, Proc. Natl. Acad. Sci. USA, 93, 6600-6604, June 1996. The optimal Tcm PKS is a complex consisting of the TcmJKLMN proteins. It is the integrity of this complex that maximizes the efficiency for the synthesis of aromatic polyketides from acetyl- and malonyl-CoA. 5. Polyketides (Dayrit)

The various Kingdoms exhibit different characteristics of their PKS enzymes. In the microbial kingdom, at least three types of PKS enzymes have been recognized. 5. Polyketides (Dayrit)

Reduction and alkylation of the polyketide chain before cyclization Reduction and alkylation of the polyketide chain before cyclization. The polyketide can be reduced to the alcohol and be subsequently dehydrated to produce the double bond. The resulting aromatic ring will not have a OH substituent in the particular position. 5. Polyketides (Dayrit)

Reduction and alkylation of the polyketide chain before cyclization Reduction and alkylation of the polyketide chain before cyclization. The polyketide can be C-alkylated (e.g., with methyl or isopentyl groups) prior to cyclization although it may be difficult to determine whether C-alkylation is carried out before or after cyclization. 5. Polyketides (Dayrit)

5. Polyketides (Dayrit)

It is used for the treatment of gram-positive bacterial infections. Erythromycin, first isolated from Streptomyces erythreus from soil samples from Iloilo sent by Abelardo Aguilar in 1949. It was first marketed by Eli Lilly as Ilosone®. R.B.Woodward accomplished its stereospecific synthesis in 1981. It is used for the treatment of gram-positive bacterial infections. 5. Polyketides (Dayrit)

5. Polyketides (Dayrit)

5. Polyketides (Dayrit)

Nature of starting unit 5. Polyketides (Dayrit)

Nature of starting unit 5. Polyketides (Dayrit)

Metabolites from polyketides The polyketide metabolites can be classified into five groups: Phenols Quinones Aflatoxins Tetracyclines Macrolide antibiotics Aromatic compounds 1. Phenols Cyclization and aromatization of polyketides form phenols as the initial product. In plants however, phenols are also formed from the shikimate pathway. Therefore, phenols and their methylated derivatives are common natural products. Some common phenols are formed via different pathways. 5. Polyketides (Dayrit)

Metabolites from polyketides 2. Quinones Quinones often occur as the final product from a series of oxidation reactions on mono- or polycyclic aromatic ring systems. The biosynthetic pathway differs in microorganisms and plants. In microorganisms, quinones arise predominatly via the polyketide pathway. In plants, however, quinones can arise via the polyketide or shikimate pathways and sometimes via the mixed biosynthetic route involving the ring-formation of an added terpenoid unit. The presence of multiple pathways to the quinone ring system may reflect the importance of this type of functionality. 5. Polyketides (Dayrit)

Overview of biosynthesis of quinones Overview of biosynthesis of quinones. Depending on the organism, quinones can arise via the polyketide or shikimate pathways. Aromatic metabolites in microorganisms are likely to be formed via the polyketide pathway while aromatic compounds in plants are likely to come from the shikimate pathway.

Metabolites from polyketides 1,4-Benzoquinone 1,4-Benzoquinone itself is the simplest member of this group. However, because it is toxic, it is not found in this form but rather as a protected precursor, such as arbutin, a glycosylated 1,4-hydroquinone, the reduced form of 1,4-benzoquinone. Arbutin occurs in the leaves of various plant species and may be a plant defense compound. The ability to detoxify phenols or to store them as glycosides appears to be a common characteristic of plants. 5. Polyketides (Dayrit)

5. Polyketides (Dayrit)

Para-quinone is a toxic compound which various organisms use. A. Various trees secrete a precursor (arbutin) to “clear” its surroundings of competing plants; B. The bombardier beetle produces para-quinone in its collecting bladder from para-hydroquinone + H2O2. 5. Polyketides (Dayrit)

5. Polyketides (Dayrit)

Metabolites from polyketides Aflatoxins The aflatoxins are a group of fungal metabolites which have closely similar chemical structures, the most evident feature being two fused furan rings. Aflatoxins were first discovered following investigations into the deaths of turkeys after being being fed mouldy peanuts. 5. Polyketides (Dayrit)

Metabolites from polyketides Aflatoxins Aflatoxins are among the most toxic naturally-occuring compounds known. They are potent hepatocarcinogens and cause lesions in the mammalian liver. They are toxic to rats down to a dose level of 1 g/day. Various strains of Aspergillus produce aflatoxins, in particular, A. parasiticus, A. versicolor and A. flavus. Aspergillus fungi are usually encountered growing on various types of organic matter, especially in damp places. They cause the decay of many stored fruits and vegetables, bread, leather goods and various fabrics. Aflatoxins are one of the major causes of concern in our copra industry. The European Commission limit is currently set at 5 ppb. 5. Polyketides (Dayrit)

Aflatoxins make up a family of polyketide metabolites Aflatoxins make up a family of polyketide metabolites. The very complex biosynthesis of aflatoxins was elucidated by George Büchi. 5. Polyketides (Dayrit)

5. Polyketides (Dayrit)

5. Polyketides (Dayrit)

5. Polyketides (Dayrit)

5. Polyketides (Dayrit)

5. Polyketides (Dayrit)

Biosynthesis of tetracyclines from Streptomyces species. 5. Polyketides (Dayrit)

Biosynthesis of tetracyclines from Streptomyces species. 5. Polyketides (Dayrit)

5. Polyketides (Dayrit)

Summary FAS and PKS probably share an evolutionary history. Like the fats, polyketides also arise from polymerization of acetyl CoA. The key features and steps are: Alternative starter units are used, in particular in the formation of tetracyclic antibiotics and macrocylic lactones. No reduction of the carbonyls, or reduction to alcohol level only. Cyclization via Claisen displacement or aldol reaction. There are many modes of cyclization depending on the chain length. 5. Polyketides (Dayrit)

Summary Aromatization often follows with loss of H2O. wider range of compounds are produced: macrocyclic lactones, phenols, quinones, and polycylic aromatic compounds. Polyketides are attractive research targets because of their strong and varied biological activity, the modular nature of the genetic system and polyketide synthases, and relatively accessible biosynthetic expression systems. 5. Polyketides (Dayrit)