Polyketides acetate units

The ansa-chain of the ansamycins streptovaricins (4), rifamycins (263), geldanamycin (4), and herbimycin (32) has been shown to be polyketide in origin, being made up of propionate and acetate units with the 0-methyl groups coming from methionine. The remaining aromatic C N portion of the ansamacroHdes is derived from 3-amino-5-hydroxybenzoic acid (264—266) which is formed via shikimate precursors. Based on the precursors of the rifamycins and streptovaricins isolated from mutant bacteria strains, a detailed scheme for the biosynthesis of most of the ansamacroHdes has been proposed (95,263). 

The framework of coccinelline-type alkaloids may be generated by linear combination of seven acetate units, as illustrated in Scheme 57 (330,336). An intermediate such as 456 would explain the existence of the different ladybug alkaloids. Support for the polyketide origin has been provided by feeding experiments ( " CHjCOONa and CHg COONa) with Coccinella septempunctata (330). 

PKSs are characterized by their ability to catalyze the formation of polyketide chains from the sequential condensation of acetate units from malonate thioesters. In plants they produce a range of natural products with varied in vivo and pharmacological properties. PKSs of particular note include acridone synthase, bibenzyl synthase, 2-pyrone synthase, and stilbene synthase (STS). STS forms resveratrol, a plant defense compound of much interest with regard to human health. STS shares high sequence identity with CHS, and is considered to have evolved from CHS more than once. ° Knowledge of the molecular structure of the CHS-like enzymes has allowed direct engineering of CHS and STS to alter their catalytic activities, including the number of condensations carried out (reviewed in Refs. 46, 51, 52). These reviews also present extensive, and superbly illustrated, discussions of CHS enzyme structure and reaction mechanism. 

Cytochalasins.—Previous results have shown that the cytochalasins, e.g. cyto-chalasin B (126), are partially polyketide in origin cf. Vol. 7, p. 29 Vol. 6, p. 44). It has now been shown that [2-2H3,2-13C]acetic acid is incorporated into cyto-chalasin B (126) in Phoma exigua and into cytochalasin D in Zygosporium masonii. Labelling of the expected sites by 13C was observed for both metabolites, but, in keeping with results on other acetate metabolites, most of the deuterium was lost, being retained only in the polyketide chain at C-l 1, which is part of the starter acetate unit.102... 

Dissection of the chemical structure of jamaicamides A-C led to the speculation that these metabolites derive from a mixture of polyketides (nine acetate units), amino acids (t-Ala and p-Ala), and the S-methyl group of methionine. To map out the biosynthetic subunits of these molecules, isotopically labeled precursors were supplied to I. majuscula JHB, and the labeling patterns discerned by NMR spectroscopy (Figure 6.12). From these experiments, insights were gained into the biochemical transformations that produce the jamaicamides, especially the mechanism of formation of the vinyl chloride group [157]. 

Stilbenoids are derived from cinnamic acid and three acetate units from mal-onyl coenzym A. The first part of the biosynthesis is in common to flavonoids. The two biosynthetic routes are diverging at the point of cyclization of a styryl-3,5,7-triketoheptanoic acid. A C-acylation produces a chalcone and subsequent modifications lead to the flavonoids. An aldol condensation of the same intermediate polyketide produces a stilbene-2-carboxylic acid that is unstable and constitutes a range of structures known as stilbenoids. Figure 9C.5 shows an overview of the biosynthetic pathway (Gorham 1995). 

Pinidine and Coniine.—Pinidine (8) and the hemlock alkaloid coniine (9) are unusual among simple piperidine alkaloids in being derived exclusively from acetate units. The combination is in each case a simple linear one and proceeds either via polyketide intermediates or, arguably in the case of coniine, via the fatty acid (10). Strong evidence from tracer and enzyme studies points to (11) as an intermediate... 

Amongst the earliest applications of labelling was the use of doubly labelled [1, 2- C2] acetate to trace the mode of incorporation of intact acetate units into a wide range of metabolites. This has been one of the major recent developments in biosynthetic methodology and permits information to be obtained which would have been impossible or at best extremely difficult to obtain by classical radio-isotope labelling techniques. The basic concept can be illustrated by a model polyketide system (Fig. 1). 

Fig. 1 a-c. Simulated proton noise decoupled NMR spectra of a polyketide-derived moiety a at natural abundance b enriched from [1,2" C2] acetate c after cleavage or rearrangement of an originally intact acetate unit... 

An example of such a polyketide is cubensic acid 76 from Xylaria cubensis [115]. From an initial viewing of this structure it could be considered to derive from a mixed acetate/propionate pathway. Feeding studies however clearly demonstrate that the underlying carbon chain is derived from eleven acetate units with all of the eight methyl branches having their origin in L-methionine (Fig. 8). 

The first PKS crystal structure, that of alfolfa chalcone synthase (CHS) in 1999 (6), had previously revealed foe type III PKS internal active site cavity and conserved Cys/His/Asn catalytic triad responsible for starter substrate loading and iterative polyketide extension (6). CHS provides the first committed chemical intermediate in flavonoid biosynthesis by catalyzing foe sequential decarboxylative addition of three acetate units from maionyl-CoA to a p-... 

Many Penicillium and Aspergillus species incorporate labelled acetate units into their polyketide metabolites sufficiently well to substantiate these biosynthetic proposals. Unfortunately, the nature of the polyketide synthase... 

C]acetate so that, when the polyketide chain was assembled, there was a chance that two labelled units, one from [l- C]acetate and the other from [2- C]acetate, would adjoin each other. The resultant metabolite would show a coupling from these adjacent labelled units. The C-3 of radicinin and deoxy-radicinin appeared as a doublet of doublets in the NMR spectrum, showing that it was at the junction of two polyketide chains. The results of this experiment contrast with that of feeding [l,2- C2]acetate, which gives information on the incorporation of intact acetate units. When radicinin was biosynthesized... 

Polyketide biosynthesis has been well smdied and there are several reviews regarding the general construction of these metabolites. " " Generally, polyketides are initiated with acetate (or propionate) derived from either acetyl-CoA (or propionyl-CoA) or malonyl-CoA (or methyknalonyl-CoA). The chain is extended by further addition of acetate units by a ketosynthase and acyltransferase domain. The acetate units can then be altered by the inclusion of a variety of domains within the PKS pathway. Common examples of alterations are ketoreductase,... 

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