Heterologous expression, isolation, and characterization of versicolorin B synthase from Aspergillus parasiticus. A key enzyme in the aflatoxin B1 biosynthetic pathway.
Aflatoxin B1 is a potent environmental carcinogen produced by certain strains of Aspergillus. Central to the biosynthesis of this mycotoxin is the reaction catalyzed by versicolorin B synthase (VBS) in which a racemic substrate, versiconal hemiacetal, is cyclized to an optically active product whose absolute configuration is crucial to the interaction of aflatoxin B1 with DNA. Attempted over-production of VBS in Escherichia coli led principally to protein aggregated into inclusion bodies but also small amounts of soluble but catalytically inactive enzyme. Comparisons to wild-type VBS by SDS-polyacrylamide gel electrophoresis and after N-glycosidase F treatment revealed that extensive glycosylation accounted for the mass discrepancy (7,000+/-1,500 Da) between the native and bacterially expressed proteins. Several over-expression systems in Saccharomyces cerevisiae were surveyed in which one that incorporated a secretion signal was found most successful. VBS of indistinguishable mass on SDS-polyacrylamide gel electrophoresis and kinetic properties from the wild-type enzyme could be obtained in 50-100-fold greater amounts and whose catalytic behavior has been examined. The translated protein sequence of VBS showed three potential N-glycosylation sites (Asn-Xaa-Ser/Thr) consistent with the modifications observed above and unexpectedly revealed extensive homology to the ADP-binding region prominently conserved in the glucose-methanol-choline (GMC) family of flavoenzymes. Over-production of VBS in yeast marks the first aflatoxin biosynthetic enzyme to be so obtained and opens the way to direct study of the enzymology of this complex biosynthetic pathway.
Silva
J C
JC
Department of Chemistry, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Townsend
C A
CA
D016604:Aflatoxin B1 / Q000096:biosynthesis*; D000581:Amidohydrolases / Q000378:metabolism; D000880:Anthraquinones / Q000378:metabolism; D001230:Aspergillus / Q000201:enzymology*; D001572:Benzofurans / Q000494:pharmacology; D015153:Blotting, Western; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010957:Plasmids / Q000378:metabolism; D012441:Saccharomyces cerevisiae
D000880:Anthraquinones; D001572:Benzofurans; D016604:Aflatoxin B1; D002265:Carboxylic Ester Hydrolases; C062670:versiconal hemiacetal acetate converting enzyme; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; C029827:versicolorin C