Characterization of SafC, a catechol 4-O-methyltransferase involved in saframycin biosynthesis.
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 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) = approximately 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.
Nelson
James T
JT
Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
Lee
Jaeheon
J
Sims
James W
JW
Schmidt
Eric W
EW
D002394:Catechol O-Methyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007546:Isoquinolines / Q000378:metabolism; D007700:Kinetics; D007980:Levodopa / Q000378:metabolism; D015394:Molecular Structure; D016941:Myxococcus xanthus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D007546:Isoquinolines; D011994:Recombinant Proteins; C057297:saframycin Mx1; D007980:Levodopa; D002394:Catechol O-Methyltransferase