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Applied and Environmental Microbiology, June 2007, p. 3575-3580, Vol. 73, No. 11
0099-2240/07/$08.00+0     doi:10.1128/AEM.00011-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of SafC, a Catechol 4-O-Methyltransferase Involved in Saframycin Biosynthesis

James T. Nelson,^1, Jaeheon Lee,^2, James W. Sims,¹ and Eric W. Schmidt^1,2*

Department of Medicinal Chemistry,¹ Department of Biology, University of Utah, Salt Lake City, Utah 84112²

Received 3 January 2007/ Accepted 10 April 2007

Members of the saframycin/safracin/ecteinascidin family of peptide natural products are potent antitumor agents currently under clinical development. Saframycin MX1, from Myxococcus xanthus, is synthesized by a nonribosomal peptide synthetase, SafAB, and an O-methyltransferase, SafC, although other proteins are likely involved in the pathway. SafC was overexpressed in Escherichia coli, purified to homogeneity, and assayed for its ability to methylate a variety of substrates. SafC was able to catalyze the O-methylation of catechol derivatives but not phenols. Among the substrates tested, the best substrate for SafC was L-dihydroxyphenylalanine (L-dopa), which was methylated specifically in the 4'-O position (k_cat/K_m = 5.5 x 10³ M^–1 s^–1). SafC displayed less activity on other catechol derivatives, including catechol, dopamine, and caffeic acid. The more labile L-5'-methyldopa was an extremely poor substrate for SafC (k_cat/K_m = 2.8 x 10^–5 M^–1 s^–1). L-Dopa thioester derivatives were also much less reactive than L-dopa. These results indicate that SafC-catalyzed 4'-O-methylation of L-dopa occurs prior to 5'-C-methylation, suggesting that 4'-O-methylation is likely the first committed step in the biosynthesis of saframycin MX1. SafC has biotechnological potential as a methyltransferase with unique regioselectivity.

Published ahead of print on 20 April 2007.

J.T.N. and J.L. contributed equally to this work.

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