MtmMII-mediated C-Methylation during Biosynthesis of the Antitumor Drug Mithramycin Is Essential for Biological Activity and DNA-Drug Interaction

Abstract

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 ␣-amino domain and an ␣/␤-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.

Functional Identification of Triterpene Methyltransferases from Botryococcus braunii Race B

“…However, C-methylation of specialized metabolites is much less common than O-or N-methylation reactions. The polyketides oxytetracycline and mithramycin from Streptomyces rimosus and Streptomyces argillaceus, for instance, are C-methylated (45,46), as is geranyl diphosphate by Streptomyces lasaliensis, which produces the soil-like odor methyl isobutyl alcohol from this methylated precursor (47). The triterpene methyltransferases described here represent a third example of C-specific methyltransferases that contribute to the biosynthesis of specialized compounds serving a unique physiological role, but in a eukaryote rather than a prokaryote system.…”

Metal–Organic Coordination versus Hydrogen Bonding: Highly Efficient Templated Photocycloadditions of Trisubstituted Isomeric Olefins in the Solid State

“…Mithramycin). [27] Whereas the reactivity of trisubstituted olefins is well-documented in solution, such molecules remain largely unexplored as reactants in solids. [15] Solid-state photodimerizations of trisubstituted olefins have been generally limited to retinoids, [28][29] coumarins, [30] quinones, [31] and substituted cycloalkenes.…”

Catalytic role of the C-terminal domains of a fungal non-reducing polyketide synthase

“…Methylation could occur either during extension when a b-ketothiolester would act as a nucleophile to attack SAM provided by the CMeT domain, or alternatively the nucleophile could be the electron-rich aromatic ring of an orsellinate intermediate (Scheme 1). Both pathways have precedents: methylation of b-ketothiolesters occurs during the biosynthesis of highly reduced (hr) polyketides such as dihydromonacolin L by the lovastatin nonaketide synthase (LNKS); 12 while C-methylation of electron rich aromatics is known during the biosynthesis of novobiocin 13 and mithramycin 14 for example. Sequence analysis of the MOS CMeT domain gives no clues about when it might act, but its location after the ACP in MOS is different from the location of CMeT domains in most other fungal PKS such as LNKS where they occur before the ACP and clearly act during chain extension.…”