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Background. The anthraquinones norsolorinic acid (NA) and averantin (AVN) accumulate in some mutant strains of Aspergillus parasiticus and are intermediates in the aflatoxin (AF) pathway (4, 8, 9, 10). Disruption of the nor-1 gene in A. parasiticus resulted in NA accumulation (5), confirming the function of Nor-1 in AF synthesis and suggesting that NA is a substrate for this protein. The nor-1 mutant also accumulates small amounts of AF, suggesting that another enzyme can bypass the step catalyzed by Nor-1. Based on nucleotide sequence data, nor-1 was proposed to encode a short-chain alcohol dehydrogenase that could convert the 1' keto group of NA to the 1' hydroxyl group of AVN (12).

Construction of pMN1, a recombinant Nor-1 expression vector. The plasmid pNOR contained a nor-1 cDNA cloned into the EcoRI/XhoI sites of pBluescript SK() (Stratagene Cloning Systems, La Jolla, Calif.). Nucleotide sequence analysis of the nor-1 cDNA was performed with a commercial product (Sequenase II; U.S. Biochemical Corp., Cleveland, Ohio). The cDNA sequence was identical to the genomic DNA sequence reported previously under accession no. L278801 except for 6 nucleotides at the 5' end of the predicted coding region. These were either not present in the mRNA or deleted during cDNA production or cloning.

Preparation of recombinant Nor-1 proteins MBP-Nor-1c and Nor-1c. pMN1 was transformed into E. coli DH5 and induced to express fusion proteins by standard methods (1). Recombinant fusion proteins (known as MBP-Nor-1c proteins) produced by cultures of selected transformants were purified by standard procedures described in the Protein Fusion and Purification system (New England Biolabs, Inc.). Two MBP-Nor-1c proteins with apparent 74- (major) and 78-kDa (minor) masses were detected in E. coli cell extracts by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Fig. 1). These proteins were not observed in extracts from noninduced cells, in control cells containing pMAL-c2, or in cells without vectors, suggesting that they are recombinant Nor-1 proteins expressed from nor-1 cDNA in pMN1. According to cDNA and genomic sequence data, MBP-Nor-1c proteins lacked the first two amino acid residues (M and T) of the Nor-1 coding sequence (12) which were replaced by eight amino acids (I, S, E, L, I, R, H, and E) from pMAL-c2 during vector construction.

View larger version (41K): [in this window] [in a new window]   FIG. 1.   Sodium dodecyl sulfate-polyacrylamide gel electrophoresis detection of MBP-Nor-1c and Nor-1c. Expression vector pMN1, containing the nor-1 cDNA fused with malE, which encodes a maltose-binding protein (MBP), was transformed into E. coli DH5. Under isopropyl--D-thiogalactopyranoside (IPTG) induction, major and minor MBP-Nor-1c fusion proteins were produced. Lanes: 1, molecular mass standard (kDa); 2, total crude extract (10,000 × g supernatant) from E. coli DH5; 3, total crude extract from E. coli DH5 transformed with expression vector pMN1; 4, MBP-Nor-1c 74-kDa (major) and 78-kDa (minor) fusion proteins, purified by affinity chromatography; 5, 32-kDa (major) and 36-kDa (minor) Nor-1c proteins and MBP (42 kDa) obtained by factor Xa cutting of the two Nor-1c-MBP fusion proteins.

View larger version (53K): [in this window] [in a new window]   FIG. 2.   Enzyme activity assay of Nor-1c-MBP and Nor-1c proteins. All reactions were conducted at 37°C in the dark. The reaction products were resolved by TLC with benzene-ethyl acetate (7:3) as the developing system. The photograph was taken under white light. The reaction components for each lane are as follows. Lane 1, NA (0.9 mM); lane 2, AVN (0.9 mM); lane 3, NA (0.9 mM) plus NADPH (2.3 mM); lane 4, NA (0.9 mM) plus NADPH (2.3 mM) plus crude cell extract (35 µg) from E. coli DH5; lane 5, NA (0.9 mM) plus NADPH (2.3 mM) plus crude cell extract (70 µg) from E. coli DH5 containing pMAL-c2; lane 6, NA (0.9 mM) plus NADPH (2.3 mM) plus crude cell extract (70 µg) from E. coli DH5 containing pMN1; lane 7, NA (0.9 mM) plus crude cell extract from (70 µg) E. coli DH5 containing pMN1; lane 8, NA (0.9 mM) plus NADPH (2.3 mM) plus crude cell extract (35 µg) from E. coli DH5 plus purified Nor-1c (35 µg); lane 9, NA (0.9 mM) plus crude cell extract (35 µg) from E. coli DH5 plus purified Nor-1c (35 µg). All crude cell extracts are the 10,000 × g supernatant fraction.

Preparation of reaction substrate NA. Nor-1, a nor-1-disrupted strain of A. parasiticus (13), was used to prepare NA as a potential substrate for Nor-1 activity. Conidia (~10⁸) of Nor-1 were cultured in 2 liters of yeast extract-sucrose liquid medium for 2 days at 29°C with constant shaking (150 rpm) in the dark; they were then incubated without shaking at 30°C in the dark for an additional 13 days. Red-colored mycelia (250 g [wet weight]) were collected by filtration and extracted three times with 300 ml of acetone. The acetone extracts were combined and dried with a Rotovapor R110 (Brinkmann Instruments, Inc., Westbury, N.Y.) at 65°C. The resulting solid was resuspended in 100 ml of chloroform which was concentrated (Rotovapor R110; 72°C) to 6 ml and extracted again with 100 ml of acetone. Purple-red NA was purified by preparative thin-layer chromatography (TLC) with PKF silica gel 60 Å chromatography plates (20 by 20 cm; Whatman, Inc., Clifton, N.J.) with chloroform-acetone (9:1) as the developing system. NA (as a brown-colored spot) was scraped from the TLC plate with a razor blade, and the NA was extracted with a small volume of chloroform.

Enzyme activity of recombinant Nor-1. The enzyme assay was conducted according to a published method (13) with modifications. The reaction mixture included protein (70 to 105 µg), NA (90 µmol), NADPH (0.23 µmol), KH₂PO₄ (90 mM [pH 7.5]), and 10% (vol/vol) glycerol in a total volume of 100 µl. Reactions were conducted at 37°C in the dark for 30 to 90 min, and stopped by addition of 900 µl of ethyl acetate. The ethyl acetate was air dried, the residue was extracted by adding 400 µl of chloroform, and the reaction products in chloroform extract were resolved by TLC (HPK silica gel 60 Å [10 by 10 cm]; Whatman, Inc.) with benzene-ethyl acetate (7:3) as the developing system (Fig. 2).

Identification of the reaction end product. Standard AVN was prepared and purified by the method of Bennett et al. (2) with modifications. A. parasiticus ATCC 56774 accumulates AVN and was used to produce this AF pathway intermediate (2). The culture conditions were the same as described above for NA purification. Orange-colored mycelia (210 g [wet weight]) were collected by filtration and extracted with acetone until colorless. Water was added (30%, vol/vol) to the combined acetone extracts, the solution was washed with hexane (1:1 ratio), and the orange pigments were extracted from the acetone phase with chloroform (1:1 ratio). The chloroform extract was concentrated to 5 ml at room temperature and the AVN was purified by preparative TLC (HPK silica gel 60 Å [20 by 20 cm]; Whatman, Inc.) with chloroform-acetone (9:1) as the developing system. AVN (as a yellow-brown-colored spot) was scraped from the TLC with a razor blade and extracted with a small volume of chloroform.

Discussion. Recombinant Nor-1c catalyzed the conversion of the 1' keto group of NA to the 1' hydroxyl group of AVN in the presence of NADPH, confirming that the protein is an NADPH-dependent NA ketoreductase (alcohol dehydrogenase) in vitro. In previous studies, disruption of nor-1 resulted in the accumulation of NA; complementation of a nor-1 mutant restored AF synthesis (13). These data support the hypothesis that native Nor-1 catalyzes the conversion of NA to AVN in A. parasiticus. Although it is clear that another enzyme can bypass the Nor-1-catalyzed step, the gene encoding this activity has not been identified. Cloning of this gene or purification of the enzyme activity should allow us to define more clearly the potential pathways for the conversion of NA to aflatoxin B₁ in the fungus.