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Applied and Environmental Microbiology, November 2000, p. 4877-4882, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Purification and Characterization of Streptomyces griseus Catechol O-Methyltransferase

Kajari Dhar and John P. N. Rosazza*

Division of Medicinal and Natural Products Chemistry, Center for Biocatalysis and Bioprocessing, College of Pharmacy, University of Iowa, Iowa City, Iowa 52242

Received 15 May 2000/Accepted 25 August 2000

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 Km 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 Ki of 224 ± 20.6 µM. Sinefungin and S-adenosylhomocysteine inhibited methylation, and the enzyme was inactivated by Hg2+, p-chloromercuribenzoic acid, and N-ethylmaleimide.

* Corresponding author. Mailing address: Division of Medicinal and Natural Products Chemistry, Center for Biocatalysis and Bioprocessing, College of Pharmacy, University of Iowa, Iowa City, IA 52242. Phone: (319) 335-8842. Fax: (319) 335-8766. E-mail: john-rosazza{at}uiowa.edu.

Applied and Environmental Microbiology, November 2000, p. 4877-4882, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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