title: Purification and characterization of Streptomyces griseus catechol O-methyltransferase.
abstract: A soluble (100,000 x g supernatant) methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to catechols was present in cell extracts of Streptomyces griseus. A simple, general, and rapid catechol-based assay method was devised for enzyme purification and characterization. The enzyme was purified 141-fold by precipitation with ammonium sulfate and successive chromatography over columns of DEAE-cellulose, DEAE-Sepharose, and Sephacryl S-200. The purified cytoplasmic enzyme required 10 mM magnesium for maximal activity and was catalytically optimal at pH 7. 5 and 35 degrees C. The methyltransferase had an apparent molecular mass of 36 kDa for both the native and denatured protein, with a pI of 4.4. Novel N-terminal and internal amino acid sequences were determined as DFVLDNEGNPLENNGGYXYI and RPDFXLEPPYTGPXKARIIRYFY, respectively. For this enzyme, the K(m) for 6,7-dihydroxycoumarin was 500 +/- 21.5 microM, and that for S-adenosyl-L-methionine was 600 +/- 32.5 microM. Catechol, caffeic acid, and 4-nitrocatechol were methyltransferase substrates. Homocysteine was a competitive inhibitor of S-adenosyl-L-methionine, with a K(i) of 224 +/- 20.6 microM. Sinefungin and S-adenosylhomocysteine inhibited methylation, and the enzyme was inactivated by Hg(2+), p-chloromercuribenzoic acid, and N-ethylmaleimide.
authors.affiliation: Division of Medicinal and Natural Products Chemistry, Center for Biocatalysis and Bioprocessing, College of Pharmacy, University of Iowa, Iowa City, Iowa 52242, USA.
authors.forename: K
authors.initials: K
authors.lastname: Dhar
authors.forename: J P
authors.initials: JP
authors.lastname: Rosazza
mesh_terms: D000595:Amino Acid Sequence; D002394:Catechol O-Methyltransferase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002396:Catechols / Q000378:metabolism; D008969:Molecular Sequence Data; D013053:Spectrophotometry / Q000379:methods; D013305:Streptomyces griseus / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity
chemical_list: D002396:Catechols; D002394:Catechol O-Methyltransferase