MtmMII-mediated C-methylation during biosynthesis of the antitumor drug mithramycin is essential for biological activity and DNA-drug interaction.
The antitumor drug mithramycin consists of a polyketide chromophore glycosylated with a trisaccharide and a disaccharide. Two post-polyketide methylations take place during mithramycin biosynthesis. One of these methylations has been shown to be very relevant for biological activity, that is the introduction of a methyl group at aromatic C-7. We have purified to 282- fold the MtmMII methyltransferase involved in this reaction. The protein is a monomer, and results from kinetic studies were consistent with a model for the enzyme acting via a compulsory order mechanism. The enzyme showed high substrate specificity and was unable to operate on structurally closely related molecules. Structural predictions suggest that the molecule is integrated by two domains, an essentially all alpha-amino domain and an alpha/beta-carboxyl domain displaying a variation of a Rossmann-fold containing the cofactor binding site. Although 7-demethyl-mithramycin did not show any biological activity, it was able to reach the nucleus of eukaryotic cells, with subsequent binding to DNA. Mithramycin and 7-demethylmithramycin were able to form similar complexes with Mg(2+), although their respective DNA binding isotherms were very different. The dinucleotide binding model fit well the isotherms recorded for both compounds, predicting that the C-7 methyl group was essential for high affinity binding to specific GC and CG sequences. Considering previous structural studies, we propose that this effect is performed by positioning the group in the floor of the minor groove, allowing the interaction with the third sugar moiety of the trisaccharide, d-mycarose, which is involved in sequence selectivity.
Rodríguez
David
D
Departamento de Biologí Funcional, Universidad de Oviedo, 33006 Oviedo, Spain.
Quirós
Luis M
LM
Salas
José A
JA
D000595:Amino Acid Sequence; D000903:Antibiotics, Antineoplastic / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology; D002467:Cell Nucleus / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008926:Plicamycin / Q000096:biosynthesis* / Q000378:metabolism / Q000494:pharmacology; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000903:Antibiotics, Antineoplastic; D004247:DNA; D008780:Methyltransferases; D008274:Magnesium; D008926:Plicamycin