A versiconal hemiacetal acetate converting enzyme in aflatoxin biosynthesis

1989 ◽  
Vol 107 (2-3) ◽  
pp. 121-126 ◽  
Author(s):  
D. P. H. Hsieh ◽  
C. C. Wan ◽  
J. A. Billington
Keyword(s):  
Converting Enzyme ◽  
Aflatoxin Biosynthesis ◽  
Versiconal Hemiacetal Acetate

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 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.

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 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.

Substrate specificity and inducibility of TACE (tumour necrosis factor α-converting enzyme) revisited: the Ala-Val preference, and induced intrinsic activity

2003 ◽  
Vol 70 ◽  
pp. 39-52 ◽  
Author(s):  
Roy A. Black ◽  
John R. Doedens ◽  
Rajeev Mahimkar ◽  
Richard Johnson ◽  
Lin Guo ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Recent Work ◽  
Tumour Necrosis Factor ◽  
Tumour Necrosis ◽  
Transforming Growth Factor ◽  
Intrinsic Activity ◽  
Converting Enzyme ◽  
Tumour Necrosis Factor Α ◽  
Factor Α ◽  
Necrosis Factor

Tumour necrosis factor α (TNFα)-converting enzyme (TACE/ADAM-17, where ADAM stands for a disintegrin and metalloproteinase) releases from the cell surface the extracellular domains of TNF and several other proteins. Previous studies have found that, while purified TACE preferentially cleaves peptides representing the processing sites in TNF and transforming growth factor α, the cellular enzyme nonetheless also sheds proteins with divergent cleavage sites very efficiently. More recent work, identifying the cleavage site in the p75 TNF receptor, quantifying the susceptibility of additional peptides to cleavage by TACE and identifying additional protein substrates, underlines the complexity of TACE-substrate interactions. In addition to substrate specificity, the mechanism underlying the increased rate of shedding caused by agents that activate cells remains poorly understood. Recent work in this area, utilizing a peptide substrate as a probe for cellular TACE activity, indicates that the intrinsic activity of the enzyme is somehow increased.

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