Structural Basis\nof Substrate Recognition by the Postmitosane\nModification Enzyme MitM in Mitomycin Biosynthesis

Mitomycins make up a class of natural molecules produced\nby <i>Streptomyces</i> with strong antibacterial and antitumor\nactivities.\nMitM is a key postmitosane modification enzyme involved in mitomycin\nbiosynthesis in <i>Streptomyces caespitosus</i>. This protein\nwas previously suggested to catalyze the aziridinium methylation of\nmitomycin A and the mitomycin intermediate 9a-demethyl-mitomycin A\nas an <i>N</i>-methyltransferase. The structural basis for\nMitM to recognize cofactor <i>S</i>-adenosyl-l-methionine\n(SAM) and substrate mitomycin A is unknown. Here, we determined the\ncrystal structures of <i>apo</i>-MitM and MitM-mitomycin\nA-<i>S</i>-adenosylhomocysteine (SAH) ternary complexes\nwith resolutions of 2.23 and 2.80 Å, respectively. We found that\nMitM adopts a class I SAM-dependent methyltransferase fold and forms\na homodimer in solution. Conformational changes in a series of residues\ninvolved in the formation of active pockets assist MitM in binding\nSAH and mitomycin A. In particular, the <sub>28</sub>ALGAASLGE<sub>36</sub> loop changes most significantly. When mitomycin A binds,\nthe bending direction of this loop is reversed, changing the entrance\nof the active site from open to closed. This study provides structural\ninsights into MitM’s involvement in the postmitosane stage\nof mitomycin biosynthesis and provides a template for the engineering\nof methyltransferases.