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

ABSTRACT 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 (kcat/Km = 5.5 × 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 (kcat/Km = ∼2.8 × 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.