Purification and characterization of versicolorin B synthase from Aspergillus parasiticus. Catalysis of the stereodifferentiating cyclization in aflatoxin biosynthesis essential to DNA interaction.

The absolute configuration of the dihydrobisfuran ring system characteristic of aflatoxin B1 is essential to the covalent reaction of its metabolically activated form with double-stranded DNA. The biosynthesis of this potent mycotoxin proceeds through three configurationally labile intermediates to racemic versiconal hemiacetal. Subsequent enzymatic cyclization establishes the stereochemistry of this, critical ring fusion in (-)-versicolorin B and is catalyzed by versicolorin B synthase (VBS). The isolation and purification of VBS from Aspergillus parasiticus (SU-1, ATCC 56775) and its kinetic characterization and attempted inactivation are described. Initial purification trials were plagued both by a chromophoric impurity which was difficult to remove and by low recoveries of active protein. The discovery of a remarkably broad pH range of enzyme stability and catalytic activity led to an efficient procedure involving preparative isoelectric focusing and ion exchange FPLC chromatography. The enzyme behaved as a dimer upon gel filtration and migrated with M(r) 78000 Da during denaturing gel electrophoresis. The UV spectrum of pure VBS gave no evidence of a bound chromophore. Detailed kinetic analysis of VBS revealed that this protein selects from two equilibrating enantiomers of versiconal hemiacetal to cyclize the appropriate antipode to optically pure versicolorin B. By varying the amount of enzyme to a fixed concentration of substrate, the rate of enzymic cyclization could be limited by the intrinsic rate of enantiomerization of the substrate under the conditions of reaction. It was possible to quantitate the dynamics of this substrate enantiomerization/cyclization process, to establish the role played by VBS, and to evaluate the significance of each to the overall biosynthesis of aflatoxin. The potential role of an acidic residue of the enzyme in catalysis was supported by analysis of the pH-rate profile of VBS and chemical labeling studies. Successful demonstration of competitive inhibition of VBS by a simple substrate analogue led to the design and synthesis of a potential mechanism-based inactivator of the protein.