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Abstract：
Metal insertion into an engineered cytoplasmic form of the multicopper enzyme N2O reductase (N2OR) (EC 1.7.99.6) of Pseudomonas stutzeri was studied. The reductase has an unusually long presequence of 50 amino acids for translocation into the periplasm. The signal peptide of N2OR shares a conserved twin-arginine sequence motif with the signal peptides of other N2O reductases and a sizeable group of periplasmic or membrane-bound enzymes, requiring cofactor insertion or processing. A catalytically inactive reductase, N2ORR20D, that lacked Cu, accumulated in the cytoplasm on mutation of the first arginine of this motif. The CuA site of N2ORR20D could be reconstituted in vitro indicating that the lack of metal was not due to a serious conformational restraint. Our findings locate the event of in vivo Cu insertion into N2OR in the periplasm or allow it to take place concomitant with protein translocation.
摘要：
研究了将金属插入Stutzeri假单胞菌的多铜酶N2O还原酶（N2OR）（EC1.7.99.6）的工程化细胞质形式。还原酶具有异常长的50个氨基酸的前序列，用于易位到周质中。N2OR的信号肽与其他N2O还原酶的信号肽和大量的周质或膜结合酶共享保守的双精氨酸序列基序，需要辅因子插入或处理。一种无催化活性的还原酶，N2ORR20D,缺乏铜，在该基序的第一个精氨酸突变时积累在细胞质中。N2ORR20D的CuA位点可以在体外重建，表明金属的缺乏不是由于严重的构象限制。我们的发现定位了体内Cu插入周质中N2OR的事件，或使其与蛋白质易位同时发生。
