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Applied and Environmental Microbiology, December 2005, p. 8963-8965, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8963-8965.2005

SHORT REPORT

An Aflatoxin Biosynthesis Cluster Gene Encodes a Novel Oxidase Required for Conversion of Versicolorin A to Sterigmatocystin

Kenneth C. Ehrlich,^* Beverly Montalbano, Stephen M. Boué, and Deepak Bhatnagar

Southern Regional Research Center/ARS/USDA, P.O. Box 19687, New Orleans, Louisiana 70124

Received 8 June 2005/ Accepted 24 August 2005

Top Abstract Introduction References

 
Disruption of the aflatoxin biosynthesis cluster gene aflY (hypA) gave Aspergillus parasiticus transformants that accumulated versicolorin A. This gene is predicted to encode the Baeyer-Villiger oxidase necessary for formation of the xanthone ring of the aflatoxin precursor demethylsterigmatocystin.

Top Abstract Introduction References

 
Some Aspergillus species produce the polyketide bisfuran metabolites versicolorin A (VA) and sterigmatocystin (ST) and the toxic and carcinogenic aflatoxins (AF) (1, 13, 15, 22). Oxidative rearrangement of VA to ST is expected to require the activity of several enzymes (2, 7, 9) encoded by genes in a biosynthetic cluster (26). A cytochrome P450 monooxygenase and a short-chain NADPH-reductase were previously shown to catalyze steps in the conversion process (12, 13, 25). We now report that the aflatoxin biosynthesis cluster gene aflY encodes an enzyme that is predicted to catalyze the Baeyer-Villiger oxidation of a dienone intermediate formed by epoxidation of the anthraquinone ring of VA.

To determine the function of aflY, a disruption vector, pAFLY, was constructed so that a 916-bp (nucleotides 68173 to 69108) portion of the coding sequence (nucleotides 67990 to 69582) in aflY (GenBank accession number AY371490) was replaced with a 7.0-kb niaD selection cassette (Fig. 1A). The primer sets used for vector construction in pUC18 were as follows: PCR-1, 5'-AATGGTACCCAGATGAGAGAACAATCAAC (67267, KpnI) and 5'-GAGTCTAGACACACATGACCATGGATTCG (68173, XbaI); PCR-2, 5'-AATTCTAGACCTGGAAGAAGCGCACGTAG (69108, XbaI) and 5'-GAGGCGCATGCTATCAACTCACGGCTTGGTATCCCA (70597, SphI). Restriction enzyme sites (underlined) and positions in aflY are in parentheses. The niaD insert used for selection of transformants was obtained by XbaI digestion of pSL82 (3). Vector construction, fungal transformation, and analysis of transformants of A. parasiticus BN009E niaD were done as previously described (6).

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FIG. 1. Preparation and characterization of aflY disruptant mutants of A. parasiticus. (A) Expected results of double-crossover insertion of pAFLY XhoI fragment into BN009E DNA. X, XhoI site. The shaded region represents the niaD cassette used as the selection marker for fungal transformation. Large arrows indicate direction of transcription. (B) Silica gel thin-layer chromatography of acetone extracts of mycelia from transformants. Lane 1, extract of mycelia from wild-type (WT) fungi. Other lanes are extracts from transformants. Plates were developed with toluene-ethylene acetate-acetic acid (80:30:4, vol/vol/vol). stds, standards. (C) Southern blot analysis of XhoI-digested DNAs from an aflY disruptant (H2, lane 2 of Fig. 1B) and untransformed BN009E with a 32P-labeled 829-bp portion of aflY as the probe. (D) RT-PCR of RNAs from hypA disruptant H2 and untransformed BN009E. Oligonucleotides used for RT-PCR hybridized to coding regions of the aflY and aflX genes.

About 10% of the A. parasiticus transformants failed to produce AFB₁ and AFG₁ but accumulated a product that comigrated with VA on thin-layer chromatography (Fig. 1B) and had the same mass spectrum (m/z = 338, 310, 309, and 281) (4). A smaller amount of a second product accumulated that was determined to be 6-deoxyVA based on its comigration with authentic material (R_f = 0.93) and its mass spectrum with ion peaks at m/z = 322, 294, 293, and 265. This material was not found in the wild type or in cultures of VAD102, a ver-1 knockout mutant (20).

Southern hybridization results showed that the niaD cassette was inserted into aflY in the putative knockout transformants (Fig. 1C). The expected 2.1-kb XhoI fragment was detected in the wild-type A. parasiticus BN009E DNA, whereas a 9.5-kb fragment was detected in transformant H2. Reverse transcription (RT)-PCR of total RNA from aflY failed to detect a transcript from H2 (Fig. 1D). Transcripts were detected for the neighboring gene aflX (ordB), indicating that only aflY was disrupted in H2.

When cultures of the aflY disruptant H2 were incubated with ST or O-methyl ST, AF was produced (Table 1). AF was not produced when either averantin, averufin (earlier precursor metabolites), or VA was fed to the cultures. Cocultivation of H2 with the ver-1 disruptant VAD102 (16) restored AF production (Table 1), indicating that it can compensate for the defect in the aflY knockout culture. However, incubation with a mycelial extract of VAD102 failed to restore AF production, suggesting that either the necessary precursor metabolite was not formed in sufficient amounts, was not sufficiently stable to survive the extraction conditions, or was not taken up by the mycelia during incubation.

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TABLE 1. AF production by A. parasiticus H2 (aflY mutant) by precursor feeding and cocultivation

Henry and Townsend proposed that reaction steps in the conversion of VA to dimethyl ST are most consistent with the following order: oxidation-reduction-oxidation (9). Such a reaction sequence is consistent with the three types of enzymes now proven to be involved in the VA-to-ST conversion process. An explanation of why mutation of any one of these genes gives a fungal isolate that mainly accumulates VA is as follows. The first step in the conversion process is predicted to be cytochrome P450 monooxygenase (StcS/VerA)-catalyzed epoxidation of the B ring of VA to give structure I (Fig. 2). This intermediate is predicted to rearrange to the dienone intermediate (structure II). Therefore, stcS/verA mutation would be expected to lead to accumulation of VA. Ver-1 is similar to T₄HN reductase, which catalyzes deoxygenation of tetrahydronaphthalene in melanin biosynthesis (23). Assuming that Ver-1-catalyzed deoxygenation is the second step in the conversion process, the dienone in Ver-1-defective mutants could revert to VA by acid-catalyzed dehydration. In aflY mutants, where Ver-1 is functional, the products formed by Ver-1-catalyzed reduction of the dienone (III, R = OH and R = H) could revert to VA and 6-deoxyVA, respectively, by dehydration. This hypothesis is consistent with the isolation of 6-deoxyVA in extracts of the aflY, but not the ver-1, knockout cultures.

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FIG. 2. Possible pathway for conversion of VA to dimethyl ST. Structures in brackets are hypothetical intermediates as proposed by Henry and Townsend (9).

A BLASTP search of the GenBank database with the predicted 495-amino-acid AflY protein (AAS66025) identified 10 putative homologs, all from fungi (accession numbers: EAA61597.1, Aspergillus nidulans StcR; EAA31946.1, Neurospora crassa; EAA53187.1, Magnaporthe grisea; EAA77237.1, Gibberella zeae; EAA62097.1, A. nidulans; EAA66025.1, A. nidulans; EAK85907.1, Ustilago maydis; EAA69522.1, G. zeae; CAG81376.1, Yarrowia lipolytica; EAL23380.1, Cryptococcus neoformans). Known conserved protein domains were not detected by the BLAST search. However, certain regions were highly conserved in most of the putative homologs. These include G₇₇FH(N/D)HxxH(H/Q₇₈), G₁₇₄x(L/V)HP(L/I/V)I(H/N/Q) (L/I)xxxxE₁₈₇, D₃₁₂FxxxH₃₁₇, D₄₀₁DGHxxKxxRA₄₁₁, and W₄₇₁VRWCG(E/D)xAW₄₈₀ (invariant amino acids are in boldface; x = any amino acid). StcR, the ST biosynthetic homolog and closest match to AflY, is 47% identical. The closely spaced His residues and other well-conserved His residues in StcR and AflY share a resemblance to His-containing sites in metallo-oxygenases that are necessary for catalysis of non-cytochrome P450 oxygen insertion into aromatic rings. Enzymes such as laccases (8, 11, 17, 19), polyphenol oxidases (24), and quercetin 2,3-dioxygenase (21) have related motifs. The short Trp-rich region near the C-terminal end may be a hydrophobic pocket that could facilitate the tethering of the bis-furan portion of the VA substrate (10, 14). The conserved Asp_401,402, Lys₄₀₇, and Arg₄₁₀ residues could help to tether a hydroxyl or keto residue during the proposed B-ring rearrangement. The first third of the protein also contains three well-conserved Tyr residues (Tyr_96,133,156) that could be required for NAD binding (5) or serve as proton donors in the rearrangement of a possible lactone intermediate (18). Therefore, this novel protein contains catalytic regions consistent with its functioning as the Baeyer-Villiger oxidase in the conversion of VA to ST.

 
* Corresponding author. Mailing address: SRRC/ARS/USDA, 1100 R. E. Lee Blvd., P.O. Box 19687, New Orleans, LA 70179. Phone: (504) 286-4369. Fax: (504) 286-4419. E-mail: ehrlich{at}srrc.ars.usda.gov.

Top Abstract Introduction References

 

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Applied and Environmental Microbiology, December 2005, p. 8963-8965, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.8963-8965.2005

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