Isolation and characterization of the versicolorin B synthase gene from Aspergillus parasiticus. Expansion of the aflatoxin b1 biosynthetic gene cluster.
Versicolorin B synthase catalyzes the side chain cyclization of racemic versiconal hemiacetal to the bisfuran ring system of(-)-versicolorin B, an essential transformation in the aflatoxin biosynthetic pathway of Aspergillus parasiticus. The dihydrobisfuran is key to the mutagenic nature of aflatoxin B1. The protein, which shows 58% similarity and 38% identity with glucose oxidase from Aspergillus niger, possesses an amino-terminal sequence homologous to the ADP-binding region of other flavoenzymes. However, this enzyme does not require flavin or nicotinamide cofactors for its cyclase activity. The 643-amino acid native enzyme contains three potential sites for N-linked glycosylation, Asn-Xaa-Thr or Asn-Xaa-Ser. The cDNA and genomic clones of versicolorin B synthase were isolated by screening the respective libraries with random-primed DNA probes generated from an exact copy of an internal vbs sequence. This probe was created through polymerase chain reaction by using nondegenerate polymerase chain reaction primers derived from the amino acid sequences of peptide fragments of the enzyme. The 1985-base genomic vbs DNA sequence is interrupted by one intron of 53 nucleotides. Southern blotting, nucleotide sequencing, and detailed restriction mapping of the vbs-containing genomic clones revealed the presence of omtA, a methyltransferase active in the biosynthesis, 3.3 kilobases upstream of vbs and oriented in the opposite direction from vbs. The presence of omtA in close proximity to vbs supports the theory that the genes encoding the aflatoxin biosynthetic enzymes in A. parasiticus are clustered.
Silva
J C
JC
Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Minto
R E
RE
Barry
C E
CE
Holland
K A
KA
Townsend
C A
CA
D016604:Aflatoxin B1 / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D000880:Anthraquinones / Q000378:metabolism*; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D005810:Multigene Family; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D000880:Anthraquinones; D017931:DNA Primers; D018076:DNA, Complementary; D004271:DNA, Fungal; D016604:Aflatoxin B1; D002265:Carboxylic Ester Hydrolases; C062670:versiconal hemiacetal acetate converting enzyme; C029827:versicolorin C