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J Biol Chem, Vol. 275, Issue 5, 3065-3074, February 4, 2000

Characterization of Two Polyketide Methyltransferases Involved in the Biosynthesis of the Antitumor Drug Mithramycin by Streptomyces argillaceus*

M. José Fernández LozanoDagger §, Lily L. Remsing, Luis M. QuirósDagger , Alfredo F. BrañaDagger , Ernestina FernándezDagger , César SánchezDagger , Carmen MéndezDagger , Jürgen Rohrpar , and José A. SalasDagger **

From the Dagger  Departamento de Biología Funcional e Instituto Universitario de Oncología, Universidad de Oviedo, 33006 Oviedo, Spain and the  Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425-2303

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 M3Delta MI mutant (mtmMI-minus mutant) accumulated 4-demethylpremithramycinone (4-DPMC) which lacks the methyl groups at carbons 4 and 9. The M3Delta M2 (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 [3H]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.

* This work was supported in part by Plan Nacional en Biotecnologia Grant BIO97-0771 (to J. A. S.), European Union Grant BIO4-CT96-0068 (to J. A. S. and J. R.), and the Medical University of South Carolina Institutional Research Funds of 1999-2000 (to J. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of a predoctoral fellowship of the Spanish Ministery of Education and Science (F.P.I.).

par To whom correspondence for chemical communications should be addressed. E-mail: rohrj@musc.edu.

** To whom correspondence for molecular biology communications should be addressed. Tel. and Fax: 34-985-103652; E-mail: jasf@sauron.quimica.uniovi.es.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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