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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >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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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

机译：丝裂霉素C结合蛋白（MRD）的醌还原酶活性的表征：从药物激活酶到药物结合蛋白的功能转换

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摘要

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-diami- nomitosene, 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-peroxi- dases. 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 en- zyme into the drug-binding component of the MC export system.
机译：产生丝裂霉素C（MC）的微生物链霉菌链霉菌中的自我保护包括MRD。一种在NADH存在下与MC结合的蛋白质，并作为独特的药物结合输出系统的组成部分起作用。 MRD的特征表明，它可以将MC还原性转化为1,2-顺式-1-羟基-2,7-二亚氨基丝氨酸，这是一种在还原性MC激活级联反应中产生的化合物。然而，天然MRD催化的还原反应是缓慢的，并且MC和还原产物都在相对较长的时间内与MRD结合。基因改组实验产生了一种突变蛋白（MRD_E55G），当在大肠杆菌中表达时，该蛋白赋予MC抗性2倍的增长。纯化的MRD_E55G还原MC的速度是天然MRD的两倍，从而生成三种与还原激活MC产生的化合物相同的化合物。详细的氨基酸序列分析表明，MRD中E55周围的区域非常类似于原核过氧化氢酶过氧化物酶的第二个活性位点。然而，天然MRD在对应于催化组氨酸的位置和在过氧化氢酶-过氧化物酶中附近的甘氨酸残基具有天冬氨酸（D52）和谷氨酸（E55）残基。突变分析表明，MRD_D52H和MRD_D52H / E55G在大肠杆菌中仅赋予MC边缘耐性。这些发现表明，MRD源自先前未确定的醌还原酶，并且MRD活性位点处的突变大大减弱了其催化活性，同时保留了底物结合能力。这种推测的进化过程可能使MRD从潜在的药物激活酶转变为MC出口系统的药物结合成分。

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