title: Purification and characterization of a methyltransferase from Aspergillus parasiticus SRRC 163 involved in aflatoxin biosynthetic pathway.
abstract: A five step scheme has been developed for the purification of a methyltransferase (MT) from mycelia of 3-day old Aspergillus parasiticus (SRRC 163), which catalyzes one step in the aflatoxin biosynthetic pathway. The S-adenosylmethionine (SAM) requiring MT activity is essential for the conversion of sterigmatocystin (ST) to O-methylsterigmatocystin (OMST) prior to being converted to aflatoxin B1. The purification of the MT was carried out from cell-free extracts by CDR (Cell Debris Remover, a cellulosic weak anion exchanger, Whatman) treatment, QMA ACELL, Hydroxylapatite-Ultrogel, PBE 94 chromatofocusing and FractoGel TSK HW-50F filtration chromatography. The purified enzyme was only about 0.1% of the total extractable proteins. The pI of the protein was about 5.0 as judged by chromatofocusing. Results of gel filtration chromatography indicated the approximate molecular mass of the native protein to be 160-KDa. SDS-polyacrylamide gel electrophoresis revealed two protein subunit bands of molecular masses approximately 110-KDa and 58-KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.87 X 10(4) M-1 cm-1 in 50 mM potassium phosphate buffer (pH 7.5). The reaction catalyzed by the MT was optimum at pH 7.5 and between 25-35 degrees C. The Km of the enzyme for ST and SAM was determined to be 1.8 microM and 42 microM, respectively with an estimated turnover number of the enzyme for ST of 2.2 X 10(-2) per sec.
authors.affiliation: Southern Regional Research Laboratory, ARS, USDA, New Orleans, Louisiana 70124.
authors.forename: D
authors.initials: D
authors.lastname: Bhatnagar
authors.forename: A H
authors.initials: AH
authors.lastname: Ullah
authors.forename: T E
authors.initials: TE
authors.lastname: Cleveland
mesh_terms: D000348:Aflatoxins / Q000096:biosynthesis*; D001230:Aspergillus / Q000201:enzymology*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004355:Drug Stability; D007700:Kinetics; D008780:Methyltransferases / Q000302:isolation & purification*
chemical_list: D000348:Aflatoxins; D008780:Methyltransferases