Abstract
A DNA chromosomal region of Streptomyces argillaceus ATCC 12596, the producer organism of the antitumor polyketide drug mithramycin, was cloned. Sequence analysis of this DNA region, located between four mithramycin glycosyltransferase genes, showed the presence of two genes (mtmMI and mtmMII) whose deduced products resembled S-adenosylmethionine-dependent methyltransferases. By independent insertional inactivation of both genes nonproducing mutants were generated that accumulated different mithramycin biosynthetic intermediates. The M3DeltaMI mutant (mtmMI-minus mutant) accumulated 4-demethylpremithramycinone (4-DPMC) which lacks the methyl groups at carbons 4 and 9. The M3DeltaM2 (mtmMII-minus mutant) accumulated 9-demethylpremithramycin A3 (9-DPMA3), premithramycin A1 (PMA1), and 7-demethylmithramycin, all of them containing the O-methyl group at C-4 and C-1', respectively, but lacking the methyl group at the aromatic position. Both genes were expressed in Streptomyces lividans TK21 under the control of the erythromycin resistance promoter (ermEp) of Saccharopolyspora erythraea. Cell-free extracts of these clones were precipitated with ammonium sulfate (90% saturation) and assayed for methylation activity using different mithramycin intermediates as substrates. Extracts of strains MJM1 (expressing the mtmMI gene) and MJM2 (expressing the mtmMII gene) catalyzed efficient transfer of tritium from [(3)H]S-adenosylmethionine into 4-DPMC and 9-DPMA3, respectively, being unable to methylate other intermediates at a detectable level. These results demonstrate that the mtmMI and mtmMII genes code for two S-adenosylmethionine-dependent methyltransferases responsible for the 4-O-methylation and 9-C-methylation steps of the biosynthetic precursors 4-DPMC and 9-DPMA3, respectively, of the antitumor drug mithramycin. A pathway is proposed for the last steps in the biosynthesis of mithramycin involving these methylation events.
Highlights
** To whom correspondence for molecular biology communications should be addressed
Two of them will modify the architecture of the polyketide moiety and involve a 4-O-methylation and a 9-C-methylation of the premithramycinone and premithramycin precursors, respectively
Experiments shown in this paper demonstrate that the products of the mtmMI and mtmMII genes are responsible for catalyzing the first two methylation reactions
Summary
D-mycarose) and a disaccharide (D-olivose-D-olivose) [18, 19]. as one of the last events in mithramycin biosynthesis, an oxidative breakage (followed by a keto reduction) of the fourth ring of the fully glycosylated intermediate premithramycin B takes place generating the final mithramycin molecule [20]. We report the cloning, sequencing, and characterization of two genes (mtmMI and mtmMII) encoding two Sadenosylmethionine-dependent methyltransferases Insertional inactivation of both genes and in vitro methylation assays demonstrated that the MtmMI and MtmMII methyltransferases are responsible for introduction of methyl groups at C-1Ј-O and C-7, respectively, of the polyketide moiety of mithramycin by Streptomyces argillaceus. These studies have allowed the identification of the possible substrates for the two methyltransferases and to propose a pathway for the methylation steps in mithramycin biosynthesis. Escherichia coli XL1-Blue [22] and E. coli XL2Blue (Stratagene) were used as hosts for subcloning. pUC18 was used for sequencing and subcloning. pIAGO2 is pWHM3 [24] containing the promoter of the erythromycin resistance gene (ermE) from Saccharopolyspora erythraea as a 0.28-kb KpnI-BamHI fragment. pBSKT is a pBluescript derivative containing a thiostrepton resistance cassette [25]. pEFBA is a pBluescript derivative containing an apramycin resistance cassette
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
