Structural Characterization of the Mitomycin 7-O- Methyltransferase MmcR Shanteri Singh 1 , Aram Chang 2 , Randal D. Goff 1 , Craig A. Bingman 2 , Sabine Grüschow 3 , David H. Sherman 3 , George N. Phillips Jr. 2,* , and Jon S. Thorson 1,* 1 Division of Pharmaceutical Sciences, Wisconsin Center for Natural Product Research, School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA 2 Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA 3 Life Sciences Institute and Department of Medicinal Chemistry, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216 Abstract Mitomycins are quinone-containing antibiotics, widely used as anti-tumor drugs in chemotherapy. Mitomycin-7-O-methyltransferase (MmcR), a key tailoring enzyme involved in the biosynthesis of mitomycin in Streptomyces lavendulae, catalyzes the 7-O-methylation of both C9β- and C9α- configured 7-hydroxymitomycins. We have determined the crystal structures of the MmcR–S- adenosylhomocysteine (SAH) binary complex and MmcR–SAH-Mitomycin A (MMA) ternary complex at resolutions of 1.9 and 2.3 Å, respectively. The study revealed MmcR to adopt a common SAM-dependent O-MTase fold and the presence of a structurally-conserved active site general acid-base pair is consistent with a proton assisted methyltransfer common to most methyltransferases. Given the importance of C7 alkylation to modulate mitomycin redox potential this study may also present a template toward the future engineering of catalysts to generate uniquely bioactive mitomycins. Keywords methyltransferase; natural product; biosynthesis; S-adenosyl-L-methionine; cancer; X-ray crystallography Introduction Originally discovered over six decades ago, 1–3 the natural product mitomycin C (MMC, Fig. 1A) remains part of the conventional anticancer regimen for solid tumors given its bias toward hypoxia-induced cytotoxicity. 4–7 Numerous bioreductants have been implicated in MMC activation via one- or two-electron reduction of the quinone, including NADPH/ cytochrome P450 reductase, DT-diaphorase and/or glutathione. Under oxygen-limiting conditions, one-electron reduction predominates, producing a highly reactive quinone methide that alkylates DNA. 4,5 However, certain MMC activating agents operate under both anaerobic and aerobic conditions (e.g., DT-diaphorase) and can thereby compromise the hypoxic selectivity of quinone-alkylating agents such as MMC. 6 The reactivity, and corresponding cytotoxic selectivity, correlates in part to the redox potential of the quinone ring which, in turn, can be modulated via appended functional groups. Modifications at the C6 and C7 positions, achieved through derivatization of naturally occurring mitomycins, * To whom correspondence should be addressed. jsthorson@pharmacy.wisc.edu; phillips@biochem.wisc.edu. NIH Public Access Author Manuscript Proteins. Author manuscript; available in PMC 2012 July 1. Published in final edited form as: Proteins. 2011 July ; 79(7): 2181–2188. doi:10.1002/prot.23040. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript