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

Namthip Sitachitta,Nicole B Lopanik,Yingqing Mao,David H Sherman

doi:10.1074/jbc.m702456200

Namthip Sitachitta, Nicole B Lopanik + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m702456200

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Abstract

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 9alpha-stereochemistry but is not involved directly in the biosynthesis of mitomycins A and C.

Highlights

Prostate, and colorectal cancer [7,8,9]
To unravel the downstream tailoring steps of mitomycin biosynthesis, we have focused on the roles of three Sadenosyl-L-methionine (AdoMet)-dependent methyltransferases identified previously in the mitomycin biosynthetic gene cluster [16]
We initially hypothesized that MitM and MitN are involved in C-9a side chain methylation for mitomycin biosynthesis

Summary

Introduction

Prostate, and colorectal cancer [7,8,9]. MC has detrimental side effects such as myelosuppression and gastrointestinal damage [10]. In the presence of MitN and AdoMet, 9-epi-MC was converted to a new product, mitomycin E [12]. The fractions that contained compound 9-epi-MC were combined, concentrated, and applied to a silica gel column chromatography followed by reverse phase HPLC to afford 0.65 mg/liter 9-epi-MC.

Results

Conclusion