Norsolorinic acid

Inhibition of aflatoxin biosynthesis by neem extracts in fungal cells appear to occur in the very early stages of the biosynthetic pathway (i.e., prior to norsolorinic acid synthesis) because after the initiation of secondary metabolism, the inhibitory effect of the neem leaf constituents was lost (84). 

Feeding experiments with isotopically labeled precursors have shown that many NR fungal polyketides are formed by the use of advanced starter units. In the classic case of norsolorinic acid 7 biosynthesis, it has long been known that hexanoate forms the starter unit. Differential specific incorporation of acetate into the early and late positions in compounds such as citrinin 3 have been used to argue that these compounds may have been formed by more than one PKS so that one PKS makes an advanced starter unit, which is passed to a second PKS for additional extension. 

Figure 34 PksA deconstruction, (a) Enzymatic domain architecture of PksA. (b) PksA utilizes a starter hexanoyl-CoA and seven malonyl-CoAs to produce the covalently linked intermediate (brackets). The PT domain acts as an aromatase/cyclase facilitating the closure of the first two rings on the intermediate. In the absence of the TE/CLC domain the intermediate undergoes C-O cyclization to spontaneously form the naphthopyrone. In the presence of the TE/CLC domain, the intermediate undergoes C-C cyclization to from the norsolorinic acid anthrone, which autooxidizes to form norsolorinic acid, (c) Observed PPant ejection ions confirming the structures of the proposed intermediates bound to the active site of the PksA T domain. The first intermediate (left) was detected on the T domain active site after incubation of SAT-KS-MAT with T domain alone. Incubation of SAT-KS-MAT with PT-T results in the formation of the intermediates containing first a single-cyclization product (middle) followed by a double-cyclization product (right).

There is considerable indirect and direct evidence that the PKS responsible for the assembly of norsolorinic acid (NSA, 79), the first isolable intermediate in the pathway to the aflatoxins, is primed by a hexanoate starter and, indeed, as discussed above,feeding [l- Cjhexanoic acid to averufin-producing cultures of Aspergillus parasiticus was reported to give some intact incorporation. To study this further we developed a method for production of NSA (79) in shake cultures and fed [2-2H3]hexanoate (Scheme 21) to these cultures in the form of the free acid (76), the ethyl ester (77) and finally the NAC thioester (78) [67]. NMR... 

Following the success of the hexanoate thioester feeding, we carried out similar experiments with H-labelled butyrate, pentanoate and heptanoate. Of these, the pentanoate results almost exactly mirrored those for hexanoate, the others showing only poor incorporation exclusively by prior degradation to acetate. The pentyl analogue (80) of norsolorinic acid was subsequently isolated by preparative hplc separation from the natural metabolite and fully characterised. Thus it appears that the NSA PKS can accept both pentanoate and hexanoate starters with comparable facility. 

Fig. 12 b. EI mass spectra of the tetramethyl ethers of norsolorinic acid (79) and the 6-fluoro-analogue (81) resulting from feeding 6-fluorohexanoic acid as its NAC thioester... 

The initial product formed by the NorS complex is predicted to be an anthrone (41). The en me required for conversion of the anthrone to the first stable intermediate in the AF biosynthesis pathway, norsolorinic acid, has not yet been identified. In A. terreus, emodin is formed from the anthrone by incorporation of molecular oxygen in a reaction catalyzed by the enzyme, emodinanthrone oxygenase (42). Putative enzymes encoded in the AF cluster... 

Figure 4. AF pathway FASs and their role in formation of norsolorinic acid anthrone. A. Domain structure of the FASs, HexA and HexB. B. Proposed complex formation for the initial steps in AF biosynthesis. Steps include hading of the HexA/HexB complex, synthesis of hexanpyl CoA, transfer ofhexanoylCoA to PksA, and iterative condensation to form the anthrone.

Figure 5. Sequence of enzymatic conversion steps in the biosynthesis of qflatoxins. A. Steps from norsolorinic acid to versicolorins. B. Steps from versicolorin A to sterigmatomcystin. C Steps from O-methylsterigmatocystin to the qflatoxins. Some of the intermediates shown are hypothetical. A question mark indicates that the enzyme suggested for the conversion step has not been proven.

Norsolorinic acid (NOR) is the first stable aflatoxin intermediate in the pathway (8, 9). The predicted conversion product from PKS in the aflatoxin... 

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