Predicted roles of the uncharacterized clustered genes in aflatoxin biosynthesis

Abstract

Biosynthesis of the toxic and carcinogenic aflatoxins (AFs) requires the activity of more than 27 enzymes. The roles in biosynthesis of newly described enzymes are discussed in this review. We suggest that HypC catalyzes the oxidation of norsolorinic acid anthrone; AvfA (AflI), the ring-closure step in formation of hydroxyversicolorone; HypB, the second oxidation step in conversion of O-methylsterigmatocystin to AF; and HypE and NorA (AflE), the final two steps in AFB(1) formation. HypD, an integral membrane protein, affects fungal development and lowers AF production while AflJ (AflS), has a partial methyltransferase domain that may be important in its function as a transcriptional co-activator.

Keywords: Aspergillus; biosynthesis; enzymes; oxidation; secondary metabolism.

Figure 1

Comparison of secondary metabolite gene clusters in six species of fungi that contain some of the genes related to AF production. The genes whose function is considered in this paper are shown above the A. flavus AF cluster schematic. Vertical lines link gene orthologs in the ST and AF clusters. Vertical red arrows are known AflR-binding sites.

Scheme 1

Formation of norsolorinic acid anthrone as the first product of polyketide biosynthesis.

Figure 2

Evidence that HypC is the norsolorinic acid anthrone (NAA) oxidase. (A) Comparison by TLC of extracts of wild type A. parasiticus BN009E and ∆hypC transformant cultures. (B) extracts of 48 hr A. parasiticus ∆hypC and ∆hypB cultures, and (C) metabolites produced by incubation of synthetic NAA with purified HypC and HypB expressed in E. coli. (NA = norsolorinic acid).

Figure 3

Alignment of HypC orthologs from fungi known to produce AF precursors or related metabolites. Putative catalytic sites are shown above and below the alignment in red text.

Scheme 2

Steps in the conversion of averufin to hydroxyversicolorone.

Scheme 3

The oxidative decarboxylation and dehydration catalyzed by the AvfA homolog OrdB (AflX) leading to xanthone ring formation in DMST.

Scheme 4

Involvement of HypB in the second oxidation step leading from OMST to the 370 Da 7-membered ring lactone precursor for AFB1 and AFG1 formation.

Scheme 5

The last steps in AFB₁ formation: HypE and NorA oxidation of putative intermediates.

Figure 4

Sequence alignment of HypE orthologs from fungi. Conserved amino acids in the EthD domain are bracketed.

Figure 5

Alignment of HypD proteins from different fungi.

Figure 6

Characteristics of A. parasiticus BN009E ΔhypD cultures compared to untransformed cultures (A) colony morphology (B) metabolite profile on TLC.

Figure 7

Alignment of AflJ orthologs from different fungi showing amino acids involved in the methyltransferase-2 domain (bracketed at bottom) and missing in the domains of AflJ orthologs from ST and AF-producing species.