Catalytic Improvement and Evolution of Atrazine Chlorohydrolase
Catalytic Improvement and Evolution of Atrazine Chlorohydrolase
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DOI：
10.1128/aem.02634-08
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发表时间：
2009-02
影响因子：
4.4
通讯作者：
C. Scott;C. Jackson;C. Coppin;R. Mourant;Margaret E. Hilton;T. Sutherland;R. Russell;J. Oakeshott
中科院分区：
文献类型：
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作者：
C. Scott;C. Jackson;C. Coppin;R. Mourant;Margaret E. Hilton;T. Sutherland;R. Russell;J. Oakeshott
阿特拉津氯水解酶AtzA在过去的50年里已经发展到催化除草剂阿特拉津的水解脱氯。它之所以引起广泛的研究兴趣，有两个原因：首先，酶的催化改进将促进其在生物修复中的应用，其次，由于它最近的发展，它提供了一个难得的机会来研究获得新的催化活性的早期阶段。利用atza -阿特拉津复合物的结构模型，将底物结合口袋的一个区域作为组合随机化的目标。通过这一过程鉴定的改进变体为AtzA酶变体的构建提供了信息，与野生型酶相比，其kcat/Km值提高了20倍。在AtzA变体中观察到Km的减少使得首次确定了AtzA催化阿特拉津脱氯的完整动力学谱，揭示了迄今未报道的AtzA底物协同性。由于底物协同性在脱氨酶中很常见，而脱氨酶是AtzA最接近的结构同源物，因此这种现象可能是AtzA进化祖先催化活性的残余。提出了一种催化机制，表明AtzA中脱氯酶活性从祖先脱氨酶进化而来的一种合理的机制途径。
ABSTRACT The atrazine chlorohydrolase AtzA has evolved within the past 50 years to catalyze the hydrolytic dechlorination of the herbicide atrazine. It is of wide research interest for two reasons: first, catalytic improvement of the enzyme would facilitate its application in bioremediation, and second, because of its recent evolution, it presents a rare opportunity to examine the early stages in the acquisition of new catalytic activities. Using a structural model of the AtzA-atrazine complex, a region of the substrate-binding pocket was targeted for combinatorial randomization. Identification of improved variants through this process informed the construction of a variant AtzA enzyme with 20-fold improvement in its kcat/Km value compared with that of the wild-type enzyme. The reduction in Km observed in the AtzA variants has allowed the full kinetic profile for the AtzA-catalyzed dechlorination of atrazine to be determined for the first time, revealing the hitherto-unreported substrate cooperativity in AtzA. Since substrate cooperativity is common among deaminases, which are the closest structural homologs of AtzA, it is possible that this phenomenon is a remnant of the catalytic activity of the evolutionary progenitor of AtzA. A catalytic mechanism that suggests a plausible mechanistic route for the evolution of dechlorinase activity in AtzA from an ancestral deaminase is proposed.