Characterization of a quinone reductase activity for the mitomycin C binding protein (MRD): Functional switching from a drug-activating enzyme to a drug-binding protein

Self-protection in the mitomycin C (MC)-producing microorganism Streptomyce s lavendulae includes MRD, a protein that binds MC in the presence of NADH and functions as a component of a unique drug binding-export system. Charac terization of MRD revealed that it reductively transforms MC into 1,2-cis-1 -hydroxy-2,7-diaminomitosene, a compound that is produced in the reductive MC activation cascade. However, the reductive reaction catalyzed by native MRD is slow, and both MC and the reduced product are bound to MRD for a rel atively prolonged period. Gene shuffling experiments generated a mutant pro tein (MRDE55G) that conferred a 2-fold increase in MC resistance when expre ssed in Escherichia coli, Purified MRDE55G reduces MC twice as fast as nati ve MRD, generating three compounds that are identical to those produced in the reductive activation of MC, Detailed amino acid sequence analysis revea led that the region around E55 in MRD strongly resembles the second active site of prokaryotic catalase-peroxidases, However, native MRD has an aspart ic acid (D52) and a glutamic acid (E55) residue at the positions correspond ing to the catalytic histidine and a nearby glycine residue in the catalase -peroxidases. Mutational analysis demonstrated that MRDD52H and MRDD52H/E55 G conferred only marginal resistance to MC in E. coli, These findings sugge st that MRD has descended from a previously unidentified quinone reductase, and mutations at the active site of MRD have greatly attenuated its cataly tic activity while preserving substrate-binding capability. This presumed e volutionary process might have switched MRD from a potential drug-activatin g enzyme into the drug-binding component of the MC export system.