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.
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.
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DOI：
10.1073/pnas.98.3.926
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发表时间：
2001-01
影响因子：
11.1
通讯作者：
Min He;P. Sheldon;D. Sherman
中科院分区：
文献类型：
--
作者：
Min He;P. Sheldon;D. Sherman
丝裂霉素C（MC）生产微生物链霉菌的自我保护包括MRD，MRD是一种在NADH存在下结合MC的蛋白质，并作为独特的药物结合-输出系统的组分发挥作用。MRD的表征表明，它将MC还原转化为1，2-顺式-1-羟基-2，7-二氨基肌醇，这是一种在还原MC活化级联中产生的化合物。然而，由天然MRD催化的还原反应是缓慢的，并且MC和还原产物都与MRD结合相对较长的时间。基因改组实验产生的突变体蛋白质（MRD（E55 G）），赋予2倍增加MC阻力时，在大肠杆菌中表达。纯化的MRD（E55 G）减少MC的速度是天然MRD的两倍，产生三种化合物，这些化合物与MC的还原活化中产生的化合物相同。详细的氨基酸序列分析表明，在MRD的E55周围的区域非常类似于原核生物过氧化氢酶-过氧化物酶的第二个活性位点。然而，天然MRD在对应于过氧化氢酶-过氧化物酶中的催化组氨酸和附近甘氨酸残基的位置处具有天冬氨酸（D52）和谷氨酸（E55）残基。突变分析表明，MRD（D52 H）和MRD（D52 H/E55 G）在E.杆菌这些发现表明MRD起源于以前未鉴定的醌还原酶，并且MRD活性位点的突变大大减弱了其催化活性，同时保留了底物结合能力。这种假定的进化过程可能已经将MRD从潜在的药物活化酶转变为MC输出系统的药物结合组分。
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.