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

Abstract

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 ( k cat / K m = 5.5 × 10 3 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 × 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.