Purification of a 40-kilodalton methyltransferase active in the aflatoxin biosynthetic pathway.

摘要

The penultimate step in the aflatoxin biosynthetic pathway of the filamentous fungi Aspergillus flavus and A. parasiticus involves conversion of sterigmatocystin to O-methylsterigmatocystin. An S-adenosylmethionine-dependent methyltransferase that catalyzes this reaction was purified to homogeneity (> 90%) from 78-h-old mycelia of A. parasiticus SRRC 163. Purification of this soluble enzyme was carried out by five soft-gel chromatographic steps: cell debris remover treatment, QMA ACELL chromatography, hydroxylapatite-Ultrogel chromatography, DEAE-Spherodex chromatography, and Octyl Avidgel chromatography, followed by MA7Q high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein peak from this step on silver staining identified a single band of approximately 40 kDa. This purified protein was distinct from the dimeric 168-kDa methyltransferase purified from the same fungal strain under identical growth conditions (D. Bhatnagar, A. H. J. Ullah, and T. E. Cleveland, Prep. Biochem. 18:321-349, 1988). The chromatographic behavior and N-terminal sequence of the 40-kDa enzyme were also distinct from those of the 168-kDa methyltransferase. The molar extinction coefficient of the 40-kDa enzyme at 278 nm was estimated to be 4.7 x 10(4) M-1 cm-1 in 50 mM potassium phosphate buffer (pH 7.5).