Purification and Characterization of Streptomyces griseus Catechol O -Methyltransferase

Abstract

ABSTRACT A soluble (100,000 × 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°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 μM, and that for S -adenosyl- l -methionine was 600 ± 32.5 μM. 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 μM. Sinefungin and S -adenosylhomocysteine inhibited methylation, and the enzyme was inactivated by Hg 2+ , p -chloromercuribenzoic acid, and N -ethylmaleimide.