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 Streptomyces 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. Characterization 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 relatively prolonged period. Gene shuffling experiments generated a mutant protein (MRD(E55G)) that conferred a 2-fold increase in MC resistance when expressed in Escherichia coli. Purified MRD(E55G) reduces MC twice as fast as native MRD, generating three compounds that are identical to those produced in the reductive activation of MC. Detailed amino acid sequence analysis revealed that the region around E55 in MRD strongly resembles the second active site of prokaryotic catalase-peroxidases. However, native MRD has an aspartic acid (D52) and a glutamic acid (E55) residue at the positions corresponding to the catalytic histidine and a nearby glycine residue in the catalase-peroxidases. Mutational analysis demonstrated that MRD(D52H) and MRD(D52H/E55G) conferred only marginal resistance to MC in E. coli. These findings suggest that MRD has descended from a previously unidentified quinone reductase, and mutations at the active site of MRD have greatly attenuated its catalytic activity while preserving substrate-binding capability. This presumed evolutionary process might have switched MRD from a potential drug-activating enzyme into the drug-binding component of the MC export system.
He
M
M
Department of Microbiology and Biological Process Technology Institute, University of Minnesota, Minneapolis, MN 55455, USA.
Sheldon
P J
PJ
Sherman
D H
DH
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001711:Biotransformation; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D026901:Membrane Transport Proteins; D016685:Mitomycin / Q000378:metabolism*; D008969:Molecular Sequence Data; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000737:chemistry / Q000378:metabolism*; D011355:Prodrugs / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D002352:Carrier Proteins; C104641:MRD protein, Streptomyces lavendulae; D026901:Membrane Transport Proteins; D011355:Prodrugs; D011994:Recombinant Proteins; D016685:Mitomycin; D016660:NAD(P)H Dehydrogenase (Quinone)