Polyketide assembly

A final crucial issue is the structure of the polyketide assembly lines. The aromatic PKSs apparently require association of their various subunits for proper functioning. In the absence of the appropriate PKS multidomain architecture, synthesis is generally inefficient and results in truncated metabolites that are subject to variable amounts of spontaneous chemistry [8,10,26], It remains unclear what minimum complement of activities is required to achieve the natural PKS products formed in vivo. A consensus is growing, however, that chain length control... 

Figure 1 Generic polyketide assembly pathway reactions catalyzed by iterative fungal polyketide synthases. The assembly sequence for the squalesatin tetraketide intermediate 37 is shown for illustration.

Two chapters in section five describe different aspects of the biosynthesis of polyketides, which are numerically the most abimdant and structurally diverse class of natural products. The first chapter reports in vivo and in vitro studies aimed at enhancing our understanding of the basic pathways of polyketide assembly with a view to producing novel compounds. In the second chapter, a consistent difference in the modes of cyclisation of the fused ring polyketides of fungi and streptomycetes is described, which provides the basis for a new biosynthetic classification of these metabolites. 

The most obvious pathway from acetate involves polyketide assembly, some sort of cyclization, oxidation of the methyl group, and oxidation of the ring. Again, you may have chosen a different order of events from ours or even a different pathway. 

While some success has been reported in analogous studies with polyketide assembly intermediates in Streptomyces metabolites, e.g. erythromycin [41] and tylosin [42], similar experiments on fungal polyketides have been more limited. The di- and tetraketide intermediates (44) and (45), variously doubly labelled with and as indicated in Scheme 14, have been incorporated into de-hydro curvular in (46) by cultures of Alternaria cineriae [43]. However, in contrast to the ease of incorporation of assembly intermediates into aspyrone by A. melleus, the experiments in A. cineriae required considerable experimentation to optimise the feeding conditions and the use of the jS-oxidation inhibitors. The initial experiments [43] depended on the use of UV mutants of A. cineriae which had lost the ability to utilise fatty acids and therefore to degrade the fatty... 

The success, albeit limited, of incorporation studies of polyketide assembly intermediates has resulted from feeding these in the form of their NAC thioesters which structurally mimic the thiol end of the phosphopantetheine moiety found in coenzyme A and the acyl carrier protein component of the PKS. This will be discussed further below, but it has also been shown that there are advantages to feeding starter units in the form of their NAC thioesters. 

For in vitro studies of polyketide assembly using the purified components of the Type II PKS, it is essential to have the ACPs in their active holo-form which can then be acylated chemically or enzymatically (Scheme 30). The inactive apo-ACPs are converted to the active holo-form by addition of phosphopantet-heine from coenzyme A to a conserved serine residue. This addition is mediated in each organism by an ACP-holo synthase. In E, coli there is an ACP-holo synthase which is part of its endogenous fatty acid biosynthetic machinery but the substrate specificity of this enzyme for heterologous ACPs has been shown... 

Many of the above studies have given invaluable information on the stereochemical outcome of the ketoreductase and dehydratase catalysed reactions occurring during polyketide assembly in fungi. A number of studies of incorporation of [2H3]acetate have provided indirect information on the stereochemistry of the final enoyl reductase reaction. Thus 2H label is found at the pro-... 

Manumycin A (52) was the first metabolite isolated from Streptomyces parvulus (strain Tii 64) [110] and its structure and absolute configuration have been described [111]. Other minor components such as manumycin B (53), C (54) and D (55) have similar structural moieties indicating their close structural and biosynthetic relationship [112]. These other compounds differ in the polyketide assembly of the acylamino side chain and in the stereochemistry at C-4. Manumycin D (55) is the first of the manumycin type compounds without an oxirane ring in the mC7N unit. Their structural elucidation has been recently carried out [112] by H NMR spectroscopy using aromatic solvent induced shift (ASIS) effects at the olefinic 3-H and circular dichroism (CD) spectroscopy has been used to determine the absolute stereochemistry of the mC7N unit. 

Rifamycin B, produced by Amycolatopsis mediterranei, is one of the most notable members of the ansamycin family [36, 37, 64, 65] (Fig. 14). It has been used clinically in a synthetically modified form called rifampicin and it is still one of the first-line therapies effective in the treatment of tuberculosis and other mycobacterial infections. The starter unit for rifamycin polyketide assembly is part of the chromophore and is derived from 3-amino-5-hydroxybenzoic acid. Five polyketide synthases are involved in the formation of rifamycin chromophore and the first polyketide synthase contains at the N terminus the loading domain for 3-amino-5-hydroxybenzoic acid, which consists of an acyl-CoA ligase linked to ACP, and module 1-3. The rifamycin polyketide synthase lacks a TE domain at the C terminus. The release of polyketide chain from polyketide synthase and the formation of amide to generate the macrocyclic lactam will be catalyzed by RifF, which is very similar to arylamine A-acetyltransferase. 

Scheme 2 Model for polyketide assembly. Putative early biosynthetic steps to the first stable pathway intermediates, akianonic acid and nogalonic acid. Structures within brackets indicate hypothetical intermediates. The numbering of the reaction steps corresponds to the numbering in Table 2, see text for discussion. Note that the carbon numbering before the polyketides are cyclized is different to the numbering of the final anthracycline end products...

Consequent upon this hypothesis, acyl intermediates would be formed that possess highly reactive poly-/3-ketone structures. Presumably, therefore, they must be stabilized on the enzyme in some way to permit chain elongation prior to cyclization and release. The manner in which this may be achieved has not been examined closely, but stabilization could probably occur after enolization of the unreduced keto groups (Packter, 1973). Binding of the resultant anions to suitable sites on the enzyme, possibly assisted by metal ion chelation (Douglas and Money, 1%7), would impose spatial restrictions on these intermediates. The position of the phenolic substituents and the nature of the ring system are therefore established at the polyketide assembly stage. 

He J, Hertweck C (2003) Iteration as programmed event during polyketide assembly molecular analysis of the aureothin biosynthesis gene cluster. Chem Biol 10 1225-1232... 

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