scholarly journals The Aspergillus parasiticus estA-Encoded Esterase Converts Versiconal Hemiacetal Acetate to Versiconal and Versiconol Acetate to Versiconol in Aflatoxin Biosynthesis

2004 ◽  
Vol 70 (6) ◽  
pp. 3593-3599 ◽  
Author(s):  
Perng-Kuang Chang ◽  
Kimiko Yabe ◽  
Jiujiang Yu
Keyword(s):  
Aspergillus Parasiticus ◽  
Enzyme Assays ◽  
Aflatoxin Biosynthesis ◽  
Cell Extracts ◽  
Esterase Gene ◽  
Versicolorin A ◽  
Versiconal Hemiacetal Acetate ◽  
Esterase Activities ◽  
Esterase Inhibitor

ABSTRACT In aflatoxin biosynthesis, the pathway for the conversion of 1-hydroxyversicolorone to versiconal hemiacetal acetate (VHA) to versiconal (VHOH) is part of a metabolic grid. In the grid, the steps from VHA to VHOH and from versiconol acetate (VOAc) to versiconol (VOH) may be catalyzed by the same esterase. Several esterase activities are associated with the conversion of VHA to VHOH, but only one esterase gene (estA) is present in the complete aflatoxin gene cluster of Aspergillus parasiticus. We deleted the estA gene from A. parasiticus SRRC 2043, an O-methylsterigmatocystin (OMST)-accumulating strain. The estA-deleted mutants were pigmented and accumulated mainly VHA and versicolorin A (VA). A small amount of VOAc and other downstream aflatoxin intermediates, including VHOH, versicolorin B, and OMST, also were accumulated. In contrast, a VA-accumulating mutant, NIAH-9, accumulated VA exclusively and neither VHA nor VOAc were produced. Addition of the esterase inhibitor dichlorvos (dimethyl 2,2-dichlorovinylphosphate) to the transformation recipient strain RHN1, an estA-deleted mutant, or NIAH-9 resulted in the accumulation of only VHA and VOAc. In in vitro enzyme assays, the levels of the esterase activities catalyzing the conversion of VHA to VHOH in the cell extracts of two estA-deleted mutants were decreased to approximately 10% of that seen with RHN1. Similar decreases in the esterase activities catalyzing the conversion of VOAc to VOH were also obtained. Thus, the estA-encoded esterase catalyzes the conversion of both VHA to VHOH and VOAc to VOH during aflatoxin biosynthesis.

scholarly journals Enzymatic Function of the Nor-1 Protein in Aflatoxin Biosynthesis in Aspergillus parasiticus

1999 ◽  
Vol 65 (12) ◽  
pp. 5639-5641 ◽  
Author(s):  
Renqing Zhou ◽  
John E. Linz
Keyword(s):  
Escherichia Coli ◽  
Aspergillus Parasiticus ◽  
Keto Group ◽  
Hydroxyl Group ◽  
Aflatoxin Biosynthesis ◽  
E Coli ◽  
Enzymatic Function ◽  
Cell Extracts ◽  
Norsolorinic Acid

ABSTRACT The nor-1 gene is involved in aflatoxin biosynthesis inAspergillus parasiticus and was predicted to encode a norsolorinic acid ketoreductase. Recombinant Nor-1 expressed inEscherichia coli converted the 1′ keto group of norsolorinic acid to the 1′ hydroxyl group of averantin in crudeE. coli cell extracts in the presence of NADPH. The results confirm that Nor-1 functions as a ketoreductase in vitro.

scholarly journals Enzymatic Conversion of Averufin to Hydroxyversicolorone and Elucidation of a Novel Metabolic Grid Involved in Aflatoxin Biosynthesis

2003 ◽  
Vol 69 (1) ◽  
pp. 66-73 ◽  
Author(s):  
Kimiko Yabe ◽  
Naomi Chihaya ◽  
Shioka Hamamatsu ◽  
Emi Sakuno ◽  
Takashi Hamasaki ◽  
...  
Keyword(s):  
Aspergillus Parasiticus ◽  
Cell Extract ◽  
Aflatoxin Biosynthesis ◽  
Cytosol Fraction ◽  
Feeding Experiments ◽  
A Cell ◽  
Molecular Masses ◽  
Microsome Fraction ◽  
Versicolorin A ◽  
Versiconal Hemiacetal Acetate

ABSTRACT The pathway from averufin (AVR) to versiconal hemiacetal acetate (VHA) in aflatoxin biosynthesis was investigated by using cell-free enzyme systems prepared from Aspergillus parasiticus. When (1′S,5′S)-AVR was incubated with a cell extract of this fungus in the presence of NADPH, versicolorin A and versicolorin B (VB), as well as other aflatoxin pathway intermediates, were formed. When the same substrate was incubated with the microsome fraction and NADPH, hydroxyversicolorone (HVN) and VHA were formed. However, (1′R,5′R)-AVR did not serve as the substrate. In cell-free experiments performed with the cytosol fraction and NADPH, VHA, versicolorone (VONE), and versiconol acetate (VOAc) were transiently produced from HVN in the early phase, and then VB and versiconol (VOH) accumulated later. Addition of dichlorvos (dimethyl 2,2-dichlorovinylphosphate) to the same reaction mixture caused transient formation of VHA and VONE, followed by accumulation of VOAc, but neither VB nor VOH was formed. When VONE was incubated with the cytosol fraction in the presence of NADPH, VOAc and VOH were newly formed, whereas the conversion of VOAc to VOH was inhibited by dichlorvos. The purified VHA reductase, which was previously reported to catalyze the reaction from VHA to VOAc, also catalyzed conversion of HVN to VONE. Separate feeding experiments performed with A. parasiticus NIAH-26 along with HVN, VONE, and versicolorol (VOROL) demonstrated that each of these substances could serve as a precursor of aflatoxins. Remarkably, we found that VONE and VOROL had ring-opened structures. Their molecular masses were 386 and 388 Da, respectively, which were 18 Da greater than the molecular masses previously reported. These data demonstrated that two kinds of reactions are involved in the pathway from AVR to VHA in aflatoxin biosynthesis: (i) a reaction from (1′S,5′S)-AVR to HVN, catalyzed by the microsomal enzyme, and (ii) a new metabolic grid, catalyzed by a new cytosol monooxygenase enzyme and the previously reported VHA reductase enzyme, composed of HVN, VONE, VOAc, and VHA. A novel hydrogenation-dehydrogenation reaction between VONE and VOROL was also discovered.

scholarly journals Function of the cypX and moxY Genes in Aflatoxin Biosynthesis in Aspergillus parasiticus

2005 ◽  
Vol 71 (6) ◽  
pp. 3192-3198 ◽  
Author(s):  
Ying Wen ◽  
Hidemi Hatabayashi ◽  
Hatsue Arai ◽  
Hiroko K. Kitamoto ◽  
Kimiko Yabe
Keyword(s):  
Gene Cluster ◽  
Rapid Method ◽  
Aspergillus Parasiticus ◽  
Cell Disruption ◽  
Aflatoxin Biosynthesis ◽  
Feeding Experiments ◽  
Phenol Extraction ◽  
Versiconal Hemiacetal Acetate

ABSTRACT The pathway oxoaverantin (OAVN) → averufin (AVR) → hydroxyversicolorone (HVN) → versiconal hemiacetal acetate (VHA) is involved in aflatoxin biosynthesis, and the cypX and moxY genes, which are present in the aflatoxin gene cluster, have been previously suggested to be involved in this pathway. To clarify the function of these two genes in more detail, we disrupted the genes in aflatoxigenic Aspergillus parasiticus NRRL 2999. The cypX-deleted mutant lost aflatoxin productivity and accumulated AVR in the mycelia. Although this mutant converted HVN, versicolorone (VONE), VHA, and versiconol acetate (VOAc) to aflatoxins in feeding experiments, it could not produce aflatoxins from either OAVN or AVR. The moxY-deleted mutant also lost aflatoxin productivity, whereas it newly accumulated HVN and VONE. In feeding experiments, this mutant converted either VHA or VOAc to aflatoxins but did not convert OAVN, AVR, HVN, or VONE to aflatoxins. These results demonstrated that cypX encodes AVR monooxygenase, catalyzing the reaction from AVR to HVN, and moxY encodes HVN monooxygenase, catalyzing a Baeyer-Villiger reaction from HVN to VHA as well as from VONE to VOAc. In this work, we devised a simple and rapid method to extract DNA from many fungi for PCR analyses in which cell disruption with a shaker and phenol extraction were combined.

scholarly journals Enzymatic Formation of G-Group Aflatoxins and Biosynthetic Relationship between G- and B-Group Aflatoxins

1999 ◽  
Vol 65 (9) ◽  
pp. 3867-3872 ◽  
Author(s):  
Kimiko Yabe ◽  
Miki Nakamura ◽  
Takashi Hamasaki
Keyword(s):  
Gene Product ◽  
Aspergillus Parasiticus ◽  
Aflatoxin Biosynthesis ◽  
Biosynthetic Activity ◽  
Free System ◽  
Cell Extracts ◽  
Enzymatic Formation ◽  
Microsome Fraction ◽  
Aflatoxin B ◽  
Cytochrome P 450

ABSTRACT We detected biosynthetic activity for aflatoxins G1 and G2 in cell extracts of Aspergillus parasiticusNIAH-26. We found that in the presence of NADPH, aflatoxins G1 and G2 were produced fromO-methylsterigmatocystin and dihydro-O-methylsterigmatocystin, respectively. No G-group aflatoxins were produced from aflatoxin B1, aflatoxin B2, 5-methoxysterigmatocystin, dimethoxysterigmatocystin, or sterigmatin, confirming that B-group aflatoxins are not the precursors of G-group aflatoxins and that G- and B-group aflatoxins are independently produced from the same substrates (O-methylsterigmatocystin and dihydro-O-methylsterigmatocystin). In competition experiments in which the cell-free system was used, formation of aflatoxin G2 from dihydro-O-methylsterigmatocystin was suppressed whenO-methylsterigmatocystin was added to the reaction mixture, whereas aflatoxin G1 was newly formed. This result indicates that the same enzymes can catalyze the formation of aflatoxins G1 and G2. Inhibition of G-group aflatoxin formation by methyrapone, SKF-525A, or imidazole indicated that a cytochrome P-450 monooxygenase may be involved in the formation of G-group aflatoxins. Both the microsome fraction and a cytosol protein with a native mass of 220 kDa were necessary for the formation of G-group aflatoxins. Due to instability of the microsome fraction, G-group aflatoxin formation was less stable than B-group aflatoxin formation. The ordA gene product, which may catalyze the formation of B-group aflatoxins, also may be required for G-group aflatoxin biosynthesis. We concluded that at least three reactions, catalyzed by the ordA gene product, an unstable microsome enzyme, and a 220-kDa cytosol protein, are involved in the enzymatic formation of G-group aflatoxins from eitherO-methylsterigmatocystin or dihydro-O-methylsterigmatocystin.

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

2005 ◽  
Vol 71 (12) ◽  
pp. 8963-8965 ◽  
Author(s):  
Kenneth C. Ehrlich ◽  
Beverly Montalbano ◽  
Stephen M. Boué ◽  
Deepak Bhatnagar
Keyword(s):  
Aspergillus Parasiticus ◽  
Aflatoxin Biosynthesis ◽  
Versicolorin A ◽  
Cluster Gene

ABSTRACT 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.

Purification and characterization of the esterases involved in aflatoxin biosynthesis in Aspergillus parasiticus

1996 ◽  
Vol 42 (8) ◽  
pp. 804-810 ◽  
Author(s):  
Ken-Ichi Kusumoto ◽  
Dennis P. H. Hsieh
Keyword(s):  
Aspergillus Parasiticus ◽  
Specific Activity ◽  
Enzyme Stability ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Preparative Chromatography ◽  
Aflatoxin Biosynthesis ◽  
Anion Exchange Column ◽  
Versiconal Hemiacetal Acetate

The esterases from the cell-free extracts (CFEs) of Aspergillus parasiticus ATCC15517, an aflatoxin-producing strain, catalyzing the hydrolytic conversion of versiconal hemiacetal acetate (VHA) to versiconal was biochemically studied. The specific activity of the enzymes increased 2.5-fold during incubation of mycelia through 40–55 h. No metal ions were required for enzyme stability, but EDTA at 1 mM and dithiothreitol at 0.5–5 mM increased its stability. Three peaks of VHA esterase activity were resolved when the proteins in the CFEs prepared from the mycelia of different ages were separated by anion-exchange column chromatography, suggesting that at least three VHA esterases were present in the eluate of this purification step. One of these esterases extracted from the mycelia of a 55-h culture was partially purified in five steps by means of preparative chromatography and fast protein liquid chromatogaphy. The partially purified enzyme when reacted with [14C]diisopropylfluorophosphate followed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis gave a single radiolabelled band, which corresponded to a protein of 32 kDa. The molecular mass of the partially purified VHA esterase determined with gel filtration was around 60 kDa. The results suggested that the enzyme consists of two isomeric subunits.Key words: aflatoxin biosynthesis, esterase, versiconal hemiacetal acetate, Aspergillus parasiticus.

scholarly journals Role of cis-Acting Sites NorL, a TATA Box, and AflR1 in nor-1 Transcriptional Activation in Aspergillus parasiticus

2005 ◽  
Vol 71 (3) ◽  
pp. 1539-1545 ◽  
Author(s):  
Michael J. Miller ◽  
Ludmila V. Roze ◽  
Frances Trail ◽  
John E. Linz
Keyword(s):  
Transcriptional Activation ◽  
Aspergillus Parasiticus ◽  
Binding Capacity ◽  
Tata Box ◽  
Mobility Shift ◽  
Gus Reporter ◽  
Cell Extracts

ABSTRACT The transcription factor AflR is required for up-regulation of specific pathway genes involved in aflatoxin biosynthesis in the filamentous fungus Aspergillus. nor-1 encodes an early aflatoxin pathway enzyme; its promoter contains a consensus AflR binding site (AflR1). Proteins in Aspergillus parasiticus cell extracts and AflR expressed in Escherichia coli do not bind to A. parasiticus AflR1 in vitro, so it was not clear if this site was required for nor-1 expression or if other transcription factors contributed to gene regulation. In this study we defined the role of AflR1 in nor-1 expression in A. parasiticus and identified additional cis-acting sites required for maximum nor-1 transcriptional activation. Deletion and substitution of AflR1 in the nor-1 promoter in A. parasiticus nor-1::GUS reporter strains showed that this site is required for nor-1 transcriptional activation in vivo. Substitution of a putative TATA box in the nor-1 promoter resulted in nondetectable β-glucuronidase (GUS) activity, demonstrating that this TATA box is functional in vivo. We also identified a novel cis-acting site, designated NorL, between residues −210 and −238 that was required for maximum nor-1 transcriptional activation in A. parasiticus grown in liquid medium and on solid medium. Using an electrophoretic mobility shift assay, we identified a specific NorL-dependent DNA-protein complex that relies on a functional AflR, either directly or indirectly, for maximum binding capacity. Because the NorL site appears only once in the aflatoxin gene cluster, its association with the nor-1 promoter may have important implications for the overall regulatory scheme for the aflatoxin pathway.

Sign in / Sign up

Export Citation Format

Share Document