Improving the pyrophosphate-inosine phosphotransferase activity of Escherichia blattae acid phosphatase by sequential site-directed mutagenesis
Improving the pyrophosphate-inosine phosphotransferase activity of Escherichia blattae acid phosphatase by sequential site-directed mutagenesis
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DOI：
10.1271/bbb.68.1046
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发表时间：
2004-05-01
影响因子：
1.6
通讯作者：
Asano, Y
中科院分区：
文献类型：
--
作者：
Mihara, Y;Ishikawa, K;Asano, Y
埃希氏菌酸性磷酸酶/磷酸转移酶 (EB-AP/PTase) 除了其固有的磷酸酶外，还具有 C-5' 位选择性焦磷酸核苷磷酸转移酶活性。通过连续定点诱变研究了其磷酸转移酶活性的改善。通过比较较高的5'-肌苷酸(5'-IMP)生产力和较低的5'-IMP生产力的酸性磷酸酶/磷酸转移酶的一级结构，选择替代的候选残基。然后进行总共11个氨基酸的连续取代。随着取代氨基酸残基数量的增加，突变酶的5'-IMP生产率增加，EB-AP/PTase的11个突变体磷酸转移酶的活性达到与摩根氏菌AP/PTase相同的水平。该结果表明，Leu63、Ala65、Glu66、Asn69、Ser71、Asp116、Thr135和Glu136（其相关性仅通过结构分析不能直接确定）也在EB-AP/PTase的磷酸转移酶活性中发挥重要作用。
Escherichia blattae acid phosphatase/phosphotransferase (EB-AP/PTase) exhibits C-5'-position selective pyrophosphate-nucleoside phosphotransferase activity in addition to its intrinsic phosphatase. Improvement of its phosphotransferase activity was investigated by sequential site-directed mutagenesis. By comparing the primary structures of higher 5'-inosinic acid (5'-IMP) productivity and lower 5'-IMP productivity acid phosphatase/phosphotransferase, candidate residues of substitution were selected. Then a total of 11 amino acid substitutions were made with sequential substitutions. As the number of substituted amino acid residues increased, the 5'-IMP productivity of the mutant enzyme increased, and the activity of the 11 mutant phosphotransferases of EB-AP/PTase reached the same level as that of Morganella morganii AP/PTase. This result shows that Leu63, Ala65, Glu66, Asn69, Ser71, Asp116, Thr135, and Glu136, whose relevance was not directly established by structural analysis alone, also plays an important role in the phosphotransferase activity of EB-AP/PTase.