title: Purification and characterization of two versiconal hemiacetal acetate reductases involved in aflatoxin biosynthesis.
abstract: Two versiconal hemiacetal acetate (VHA) reductase activities (designated I and II), which catalyzed the reaction from VHA to versiconol acetate (VOAc) during aflatoxin biosynthesis, were purified to apparent homogeneity from the cytosol fraction of the mycelia of Aspergillus parasiticus mutant NIAH-26 through the following chromatography steps: first, fractionation with ammonium sulfate and then fractionation in succession with phenyl-Sepharose, DEAE-Sepharose, Sephacryl S-300, hydroxylapatite, and Matrex gel Green A chromatography. VHA reductase I and VHA reductase II were completely separated at the end of the DEAE-Sepharose step. The apparent molecular masses of reductase I and reductase II were estimated (by gel filtration) to be approximately 390 kDa; their denaturing molecular masses were 39- and 40-kDa, respectively (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Their pI values were 6.6 and 6.0, respectively (as determined by isoelectric focusing), and the optimal pH values were 8.0 and 9.0, respectively, although both enzymes exhibited a broad optimal pH range of between 7.5 and 9.0. The K(m) values of reductase I and reductase II for VHA were 35.4 and 25.4 muM, respectively. On the other hand, in the cell-free experiments involving either VHA reductase fraction and high-performance liquid chromatography, both (2'S)- and (2'R)-VOAc enantiomers were formed from racemic VHA and more of the 2'R isomer than the 2'S isomer was produced, indicating that the VHA reductase fractions have very similar stereospecificities to the substrate.
authors.affiliation: National Institute of Animal Health, Tsukuba, Ibaraki 305, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Matsushima
authors.forename: Y
authors.initials: Y
authors.lastname: Ando
authors.forename: T
authors.initials: T
authors.lastname: Hamasaki
authors.forename: K
authors.initials: K
authors.lastname: Yabe