Aspergillus parasiticus (from Uniprot) Versiconal hemiacetal acetate esterase; part of the gene cluster that mediates the biosynthesis of aflatoxins, a group of polyketide-derived furanocoumarins, and part of the most toxic and carcinogenic compounds among the known mycotoxins. The four major aflatoxins produced by A.parasiticus are aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2). The first step of the pathway is the conversion of acetate to norsolorinic acid (NOR) and requires the fatty acid synthase subunits aflA and aflB, as well as the PKS aflC. AflC combines a hexanoyl starter unit and 7 malonyl-CoA extender units to synthesize the precursor NOR. The hexanoyl starter unit is provided to the acyl-carrier protein (ACP) domain by the fungal fatty acid synthase aflA/aflB. The second step is the conversion of NOR to averantin (AVN) and requires the norsolorinic acid ketoreductase aflD, which catalyzes the dehydration of norsolorinic acid to form (1'S)-averantin. The norsolorinic acid reductases aflE and aflF may also play a role in the conversion of NOR to AVN. The cytochrome P450 monooxygenase aflG then catalyzes the hydroxylation of AVN to 5'hydroxyaverantin (HAVN). The next step is performed by the 5'-hydroxyaverantin dehydrogenase aflH that transforms HAVN to 5'-oxoaverantin (OAVN) which is further converted to averufin (AVF) by aflK that plays a dual role in the pathway, as a 5'-oxoaverantin cyclase that mediates conversion of 5'-oxoaverantin, as well as a versicolorin B synthase in a later step in the pathway. The averufin oxidase aflI catalyzes the conversion of AVF to versiconal hemiacetal acetate (VHA). VHA is then the substrate for the versiconal hemiacetal acetate esterase aflJ to yield versiconal (VAL). Versicolorin B synthase aflK then converts VAL to versicolorin B (VERB) by closing the bisfuran ring of aflatoxin which is required for DNA-binding, thus giving to aflatoxin its activity as a mutagen. Then, the activity of the versicolorin B desaturase aflL leads to versicolorin A (VERA). A branch point starts from VERB since it can also be converted to dihydrodemethylsterigmatocystin (DMDHST), probably also by aflL, VERA being a precursor for aflatoxins B1 and G1, and DMDHST for aflatoxins B2 and G2. Next, the versicolorin reductase aflM and the cytochrome P450 monooxygenase aflN are involved in conversion of VERA to demethylsterigmatocystin (DMST). AflX and aflY seem also involved in this step, through probable aflX-mediated epoxide ring-opening step following versicolorin A oxidation and aflY-mediated Baeyer-Villiger oxidation required for the formation of the xanthone ring ,. The methyltransferase aflO then leads to the modification of DMST to sterigmatocystin (ST), and of DMDHST to dihydrosterigmatocystin (DHST). Both ST and DHST are then substrates of the O-methyltransferase aflP to yield O-methylsterigmatocystin (OMST) and dihydro-O-methylsterigmatocystin (DHOMST), respectively. Finally OMST is converted to aflatoxins B1 and G1, and DHOMST to aflatoxins B2 and G2, via the action of several enzymes including O-methylsterigmatocystin oxidoreductase aflQ, the cytochrome P450 monooxygenase aflU, but also the NADH-dependent flavin oxidoreductase nadA which is specifically required for the synthesis of AFG1
(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Eukaryota: NE > Opisthokonta: NE > Fungi: NE > Dikarya: NE > Ascomycota: NE > saccharomyceta: NE > Pezizomycotina: NE > leotiomyceta: NE > Eurotiomycetes: NE > Eurotiomycetidae: NE > Eurotiales: NE > Aspergillaceae: NE > Aspergillus: NE > Aspergillus oryzae: NE
Warning: This entry is a compilation of different species or line or strain with more than 90% amino acid identity. You can retrieve all strain data
(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) Aspergillus parasiticus: N, E.
Aspergillus parasiticus SU-1: N, E.
Aspergillus oryzae 3.042: N, E.
Aspergillus oryzae RIB40: N, E.
Aspergillus oryzae 100-8: N, E.
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA METPFAGPWHQFVEDLGQTPCLPGKDLDSILAGWGQLAGTLATRYGFPPP DESVATEDVQLDGLWLRCYTPANATGQEPVGLYFHGGGWVMGGVKEEDGF CRVISRQCQMRLVSVEYRKAPETRYPGALNDGVSAALWAQSRYENQPLVL MGTSAGGNLAFGTALRLIDQDMADKVSGVVALAPITVHPDAVPEHLKEQY TAYEENAELTVNSRAAMQVFFDCYKAPVDDVYTSCLLHPRLLALPKVYIA ELGLDTLRDDARLMKGALDTAKVPVMYDAYPGYPHCSFMFPFKSLGEHQR TFLGGVAKAVRWMS
An 82-kb Aspergillus parasiticus genomic DNA region representing the completed sequence of the well-organized aflatoxin pathway gene cluster has been sequenced and annotated. In addition to the 19 reported and characterized aflatoxin pathway genes and the four sugar utilization genes in this cluster, we report here the identification of six newly identified genes which are putatively involved in aflatoxin formation. The function of these genes, the cluster organization and its significance in gene expression are discussed.
Title: Cloning and functional expression of an esterase gene in Aspergillus parasitcus Yu J, Chang PK, Bhatnagar D, Cleveland TE Ref: Mycopathologia, 156:227, 2002 : PubMed
Within the 80 kb aflatoxin pathway gene cluster characterized earlier, and between adhA and norA genes, we have identified an estA gene encoding an esterase from wild type strain Aspergillus parasiticus SRRC 143. The 1,500 bp genomic DNA and 945 bp cDNA sequences were determined for estA. Outside of the aflatoxin pathway gene cluster, an additional copy of the estA gene (named estA2) was also cloned from the same A. parasiticus strain. Comparison of the estA and estA2 sequences showed 9 substitutions within the 314 amino acid residues of their gene products, and no apparent defect was identified in the estA2. The estA gene is a homolog of the stcI gene identified in A. nidulans involved in the biosynthesis of sterigmatocystin and dihydro-sterigmatocystin for the conversion of versiconal hemiacetal acetate to versiconal. Reverse-transcriptase polymerase chain reaction (RT-PCR) experiments demonstrated that the estA is constitutively expressed. And only this estA gene, which is located within the aflatoxin pathway gene cluster, is expressed; no expression of the estA2 gene was detected under both aflatoxin conducive and non-conducive conditions. Possible reasons for the preferential expression of the estA over the estA2 gene have been discussed.
PksA, which initiates biosynthesis of the environmental carcinogen aflatoxin B1, is one of the multidomain iterative polyketide synthases (IPKSs), a large, poorly understood family of biosynthetic enzymes. We found that dissection of PksA and its reconstitution from selected sets of domains allows the accumulation and characterization of advanced octaketide intermediates bound to the enzyme, permitting the reactions controlled by individual catalytic domains to be identified. A product template (PT) domain unites with the ketosynthase and thioesterase in this IPKS system to assemble precisely seven malonyl-derived building blocks to a hexanoyl starter unit and mediate a specific cyclization cascade. Because the PT domain is common among nonreducing IPKSs, these mechanistic features should prove to be general for IPKS-catalyzed production of aromatic polyketides.
To help assess the potential for aflatoxin production by Aspergillus oryzae, the structure of an aflatoxin biosynthesis gene homolog cluster in A. oryzae RIB 40 was analyzed. Although most genes in the corresponding cluster exhibited from 97 to 99% similarity to those of Aspergillus flavus, three genes shared 93% similarity or less. A 257-bp deletion in the aflT region, a frameshift mutation in norA, and a base pair substitution in verA were found in A. oryzae RIB 40. In the aflR promoter, two substitutions were found in one of the three putative AreA binding sites and in the FacB binding site. PCR primers were designed to amplify homologs of aflT, nor-1, aflR, norA, avnA, verB, and vbs and were used to detect these genes in 210 A. oryzae strains. Based on the PCR results, the A. oryzae RIB strains were classified into three groups, although most of them fell into two of the groups. Group 1, in which amplification of all seven genes was confirmed, contained 122 RIB strains (58.1% of examined strains), including RIB 40. Seventy-seven strains (36.7%) belonged to group 2, characterized by having only vbs, verB, and avnA in half of the cluster. Although slight expression of aflR was detected by reverse transcription-PCR in some group 1 strains, including RIB 40, other genes (avnA, vbs, verB, and omtA) related to aflatoxin production were not detected. aflR was not detected in group 2 strains by Southern analysis.
Title: The Aspergillus parasiticus estA-encoded esterase converts versiconal hemiacetal acetate to versiconal and versiconol acetate to versiconol in aflatoxin biosynthesis Chang PK, Yabe K, Yu J Ref: Applied Environmental Microbiology, 70:3593, 2004 : PubMed
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.
An 82-kb Aspergillus parasiticus genomic DNA region representing the completed sequence of the well-organized aflatoxin pathway gene cluster has been sequenced and annotated. In addition to the 19 reported and characterized aflatoxin pathway genes and the four sugar utilization genes in this cluster, we report here the identification of six newly identified genes which are putatively involved in aflatoxin formation. The function of these genes, the cluster organization and its significance in gene expression are discussed.
Title: Cloning and functional expression of an esterase gene in Aspergillus parasitcus Yu J, Chang PK, Bhatnagar D, Cleveland TE Ref: Mycopathologia, 156:227, 2002 : PubMed
Within the 80 kb aflatoxin pathway gene cluster characterized earlier, and between adhA and norA genes, we have identified an estA gene encoding an esterase from wild type strain Aspergillus parasiticus SRRC 143. The 1,500 bp genomic DNA and 945 bp cDNA sequences were determined for estA. Outside of the aflatoxin pathway gene cluster, an additional copy of the estA gene (named estA2) was also cloned from the same A. parasiticus strain. Comparison of the estA and estA2 sequences showed 9 substitutions within the 314 amino acid residues of their gene products, and no apparent defect was identified in the estA2. The estA gene is a homolog of the stcI gene identified in A. nidulans involved in the biosynthesis of sterigmatocystin and dihydro-sterigmatocystin for the conversion of versiconal hemiacetal acetate to versiconal. Reverse-transcriptase polymerase chain reaction (RT-PCR) experiments demonstrated that the estA is constitutively expressed. And only this estA gene, which is located within the aflatoxin pathway gene cluster, is expressed; no expression of the estA2 gene was detected under both aflatoxin conducive and non-conducive conditions. Possible reasons for the preferential expression of the estA over the estA2 gene have been discussed.
Aspergillus oryzae (Yellow koji mold), Aspergillus flavus, CutL1 gene for cutinase cutinase 1 precursor (EC 3.1.1.74) (cutin hydrolase 1) (l1)