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 (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 ‫؍‬ ϳ2.8 ؋ 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.

Functional and Structural Characterization of a Cation-dependent O-Methyltransferase from the Cyanobacterium Synechocystis sp. Strain PCC 6803

“…The position specificity is strikingly different, with promiscuity toward both meta-and para-positions never observed for any of the eukaryotic enzymes. A comparable specificity has been described for the prokaryotic SafC gene product from M. xanthus when tested with caffeic acid, although the natural substrate for the latter enzyme appears to be L-dihydroxyphenylalanine, a biosynthetic precursor of the isoquinoline alkaloid saframycin (13). The precise structural features of the Synechocystis protein that contribute to this unexpected phenomenon are as yet unclear.…”

“…The conserved presence of at least one gene locus in most prokaryotes points to an important in vivo function for these organisms including Synechocystis. Sequence identities to the SafC gene product from M. xanthus methylating the saframycin precursor L-dihydroxyphenylalanine (13) as well as to the mdmC gene in Streptomyces mycarofaciens, proposed to encode the 4-O-methyltransferase for the 16-membered lactone ring of the macrolide antibiotic midecamycin (43) point to a possible role in the biosynthesis of complex secondary metabolites. In contrast to the bisquinone structure of saframycin, the midecamycin molecule gives no indication of any aromatic vicinal dihydroxy system, therefore the proposed methylation step remains questionable.…”

“…Combined use of one-and two-dimensional NMR experiments resulted in an unambiguous assignment of all 1 H and 13 C NMR signals of 3,5-dihydroxy-4-methoxycinnamic acid (with the exception of the 13 C signal of -COOH, for sensitivity reasons). Only a 4-Omethylation is in accordance with the isochronous NMR signals of positions 2/6 and 3/5.…”

Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids

“…To put this bioproduction approach into practice, detailed understanding of the biosynthetic mechanism of this class of natural products is essential. Therefore, there has been a considerable study on the subject involving gene isolation [28][29][30], substrate feeding experiments [31,32], biochemical characterizations [30,33] and bioinformatic analyses [30]. In this review, the current understanding of the mechanism of biosynthesis of 9 is described.…”

“…Molecules 2016, 21, 1078 6 of 19 [31,32], biochemical characterizations [30,33] and bioinformatic analyses [30]. In this review, the current understanding of the mechanism of biosynthesis of 9 is described.…”