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J. Biol. Chem., Vol. 282, Issue 29, 20941-20947, July 20, 2007

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Analysis of a Parallel Branch in the Mitomycin Biosynthetic Pathway Involving the mitN-Encoded Aziridine N-Methyltransferase^*

Namthip Sitachitta^**, Nicole B. Lopanik¹, Yingqing Mao^**2, and David H. Sherman^¶||**3

From the Life Sciences Institute and Departments of Medicinal Chemistry, ^¶Chemistry, and ^||Microbiology & Immunology, University of Michigan, Ann Arbor, Michigan 48109 and the ^**Microbiology and Biotechnology Institute, University of Minnesota, Minneapolis, Minnesota 55455

Mitomycin C is a natural product with potent alkylating activity, and it is an important anticancer drug and antibiotic. mitN, one of three genes with high similarity to methyltransferases, is located within the mitomycin biosynthetic gene cluster. An inframe deletion in mitN of the mitomycin biosynthetic pathway was generated in Streptomyces lavendulae to produce the DHS5373 mutant strain. Investigation of DHS5373 revealed continued production of mitomycin A and mitomycin C in addition to the accumulation of a new mitomycin analog, 9-epi-mitomycin C. The mitN gene was overexpressed in Escherichia coli, and the histidine-tagged protein (MitN) was purified to homogeneity. Reaction of 9-epi-mitomycin C with MitN in the presence of S-adenosylmethionine yielded mitomycin E showing that the enzyme functions as an aziridine N-methyltransferase. Likewise, MitN was also shown to convert mitomycin A to mitomycin F under the same reaction conditions. We conclude that MitN plays an important role in a parallel biosynthetic pathway leading to the subclass of mitomycins with 9-stereochemistry but is not involved directly in the biosynthesis of mitomycins A and C.

Received for publication, March 22, 2007 , and in revised form, May 15, 2007.

^* This work was supported by National Institutes of Health (NIH) Grant CA/GM81172 and the John G. Searle professorship (to D. H. S.). NMR instrumentation was provided with funds from the National Science Foundation (BIR-961477) and the University of Minnesota Medical School. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1 and Table 1.

¹ Supported by NIH National Research Service Award Postdoctoral Fellowship 5F32CA110636.

² Current address: Raybiotech Inc., Norcross, GA 30092.

³ To whom correspondence should be addressed: Life Sciences Inst. and Dept. of Medicinal Chemistry, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI 48109-2216. Tel.: 734-615-3129; Fax: 734-615-9907; E-mail: davidhs{at}lsi.umich.edu.

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