Molecular characterization of an Aspergillus parasiticus dehydrogenase gene, norA, located on the aflatoxin biosynthesis gene cluster

Abstract

An Aspergillus parasiticus cDNA library was screened with monoclonal antibody raised against a purified A. parasiticus 43-kDa protein demonstrating norsolorinic acid reductase (NOR) activity. One immunopositive clone contained a cDNA insert of 1,418 bp. DNA sequence analysis of this cDNA identified an open reading frame of 1,167 bp that represented the norA gene. The deduced amino acid sequence of the norA coding region consisted of 388 residues capable of encoding a polypeptide of 43.7 kDa. Southern blot analysis of genomic DNA from A. parasiticus indicated that there may be an additional copy of norA. Western blot (immunoblot) analysis of crude protein extracts of A. parasiticus mycelia demonstrated a band of reactivity at 43 kDa only when the fungus was grown in a medium conducive to aflatoxin biosynthesis. Northern (RNA) blot analysis of total RNA from the fungus demonstrated a band of hybridization at about 1.5 kb. As observed with the fungal NORA protein, the norA transcript was present only when the fungus was grown in medium conducive to aflatoxin biosynthesis. Hybridization of the norA cDNA with cosmid DNAs known to encompass a major portion of the A. parasiticus and Aspergillus flavus aflatoxin biosynthetic pathway gene cluster placed the norA gene coding region just upstream of the ver-1 gene. The deduced amino acid sequence of norA had 49% amino acid identity with that of an aryl-alcohol dehydrogenase (aad) gene from Phanerochaete chrysosporium.

Molecular biology of mycotoxin biosynthesis

“…A second reductase capable of converting NA, which shows little homology to nor1, norA, was located in the AF gene cluster of A. £avus and A. parasiticus and was cloned using monoclonal antibodies to NAR [31]. stcV in A. nidulans encodes a similar deduced product to that of norA; however, the function of stcV has not been con¢rmed [22].…”

Genetic organization and function of the aflatoxin B1 biosynthetic genes

“…In fact, a second reductase capable of converting NA to AVN in vitro was previously puri¢ed to homogeneity and shown to be a single subunit protein of about 40 kDa [21,29]. Using monoclonal antibodies against this NA-reductase, Cary et al [17] identi¢ed a cDNA clone corresponding to a gene, norA, located in the a£atoxin gene cluster of both A. parasiticus and A. £avus (Fig.…”

“…1). The predicted amino acid sequences of NOR1 and NORA share low identity (22%), but both have motifs indicating a dehydrogenase-type enzyme with adenine nucleotide-binding domains [17,28]. Attempts to disrupt both copies of the norA in a wild-type A. parasiticus strain failed [17].…”

“…The predicted amino acid sequences of NOR1 and NORA share low identity (22%), but both have motifs indicating a dehydrogenase-type enzyme with adenine nucleotide-binding domains [17,28]. Attempts to disrupt both copies of the norA in a wild-type A. parasiticus strain failed [17]. In A. nidulans, stcE and stcV have gene products with amino acid sequences similar to NOR1 and NORA, respectively; however, con¢rmation of the function of these genes is lacking [19].…”

“…£avus and A. parasiticus, and they estimated the cluster size to be 75 kb. The relative position of genes within the clusters and the nucleotide sequence of the genes are similar between the two species, although A. parasiticus appears to have an additional copy of several genes indicating that at least part of the cluster is duplicated [17,18].…”