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

Abstract

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.

Antitumor Antibiotics: Bleomycin, Enediynes, and Mitomycin

“…This NADH requirement led to the discovery that Mrd can, in fact, act as a weak activator of 19 through the Mrd-dependent generation of 1,2-cis-1-hydroxy-2,7-diaminomitosene, a compound that is produced in the reductive 19 activation cascade. 243 The finding of a 19-activating activity appeared to be conflicting with the previously identified protective effect of Mrd. However, the reductive reaction catalyzed by Mrd is slow and results in a prolonged association of 19 and its corresponding reduced product with the protein.…”

“…Further characterization revealed that Mrd was able to reversibly bind and sequester 19 with no observable antibiotic modification. 243 However, Mrd was shown to require NADH to exert its protective drugbinding function. This NADH requirement led to the discovery that Mrd can, in fact, act as a weak activator of 19 through the Mrd-dependent generation of 1,2-cis-1-hydroxy-2,7-diaminomitosene, a compound that is produced in the reductive 19 activation cascade.…”

Intramolecular Valence and Spin Interaction inmesoandracDiastereomers of ap-Quinonoid-Bridged Diruthenium Complex

“…In order to rationalize the intricate electronic interactions between transition metal ions and quinone redox systems in biochemical environments, there have been considerable investigations at the molecular level of metal complexes with O,O′-chelating o -quinonoid ligands, particularly in assigning the valence state distribution at the metal−quinone interface . However, despite the biochemical significance, , far fewer results have been reported for the coordination chemistry of p -quinonoid ligands, , including those with combined o , p -quinone functions. − The present study is aimed at exploring the electronic structural aspects of two diastereomeric diruthenium compounds, [(acac) 2 Ru(μ-L)Ru(acac) 2 ], 1 and 2 (Scheme ), where L 2− is the two-electron-reduced p -quinonoid ligand 1,4-dioxido-2,3-bis(3,5-dimethylpyrazol-1′-yl)benzene and acac − = acetylacetonate = 2,4-pentanedionate. Although 1,4-dioxido-2,5-bis(pyrazol-1′-yl)benzene, (L′) 2− , has been used previously for the formation of polynuclear complexes, − including [(bpy) 2 Ru(μ-L′)Ru(bpy) 2 ] n , the new compounds 1 and 2 represent the first set of polynuclear metal complexes bridged by L 2− .…”

“…However, para -quinones (Chart ) such as vitamin K derivatives, ubiquinones, or plastoquinones also play many important roles in energy conversion (photosynthesis, respiration) and information transfer. , …”

Sensitivity of the Valence Structure in Diruthenium Complexes As a Function of Terminal and Bridging Ligands

“…The donor substituents (O or N) at the 1,4 positions may combine with the quinone oxygen atoms to create a bis(chelate) situation with a noninnocent bridge for dimetal coordination. The combination of various accessible valence states of biochemically relevant noninnocent quinonoid systems (Q 0 = quinone, Q •– = semiquinonate, Q 2– = catecholate) and of ruthenium (Ru II , Ru III , Ru IV ) can result in complex valence and spin distribution patterns at the metal–ligand interface . Close lying and efficiently mixing frontier orbitals are responsible for this behavior, and the high degree of covalent bonding can even preclude specific oxidation state descriptions .…”