title: Purification of a 40-kilodalton methyltransferase active in the aflatoxin biosynthetic pathway.
abstract: 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).
authors.affiliation: Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, Louisiana 70179.
authors.forename: N P
authors.initials: NP
authors.lastname: Keller
authors.forename: H C
authors.initials: HC
authors.lastname: Dischinger
authors.forename: D
authors.initials: D
authors.lastname: Bhatnagar
authors.forename: T E
authors.initials: TE
authors.lastname: Cleveland
authors.forename: A H
authors.initials: AH
authors.lastname: Ullah
mesh_terms: D000348:Aflatoxins / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D008780:Methyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight
chemical_list: D000348:Aflatoxins; D008780:Methyltransferases