| Mitomycin C (MC) is an antibiotic antitumor agent produced by Streptomyces lavendulae. Upon reductive activation by intracellular NAD(P)H, MC kills cells by forming covalent cross-links in DNA. A protein produced by S. lavendulae to protect itself from MC, called MRD, functions as part of a drug export system. MRD binds to MC, and slowly catalyzes its NADH-facilitated reduction to a diaminomitosene (DAM) derivative. This dissertation describes the X-ray crystal structure of MRD both with and without DAM. Another structure with partially bound NADH is also discussed, including hypotheses about the evolutionary history of the catalytic mechanism.; The protein was subcloned, expressed, purified and crystallized. The Single-wavelength Anomalous Diffraction (SAD) method was used to determine the structure of MRD from a single seleno-methionine labeled crystal to 1.3 Å resolution. A 1.5 Å data set, containing DAM at full occupancy, was used to determine the structure of the complex, while a 1.6 Å native set was used to determine the unbound form. The complex reveals symmetry-related binding sites on either end of the dimer, with the quinone moiety of DAM engaging in π-stacking interactions between His-38 and Trp-108. The 1.3 Å (partial occupancy) data set suggests that the nicotinamide moiety of NADH competes for this His/Trp “slot.” Finally, the structure also reveals a domain-swapping event, such that there are two different forms of the dimer, representing two topologically distinct domain connectivities between the two pairs of symmetry-related α/β domains. |
