Experimental reconstruction of the molecular evolution of atrazine-degrading enzymes

酶是强大的生物催化剂，能以极高的效率和特异性加速代谢反应。尽管了解新的酶是如何从现有的酶自然进化出来的很有趣也很重要，但到目前为止，这个过程几乎没有被分析过。显然，对于能够降解仅在过去几十年才引入环境的人为物质的酶，其潜在机制可以得到最好的研究。因此，这些酶的进化一定是在这短时间内发生的。蛋白质AtzA、AtzB和AtzC提供了一个很好的例子，它们分解除草剂阿特拉津，使其宿主生物能够将反应产物用作碳和氮源。在氨基酸序列、三维结构和反应机制方面的相似性表明，ATZ酶是由氨基水解酶超家族亚型III的不同成员的基因复制和随后的专门化进化而来的，其中许多亚型是脱氨基的。根据这一假设，AtzB酶表现出低的混杂鸟嘌呤脱氨酶(GUAD)活性。在拟议的项目中，将通过合理的蛋白质工程促进这一活动，该工程基于对残基保存模式的详细比较，以确定AtzB和GUAD的不同底物特异性。相反，高AtzB活性应建立在最密切相关的鸟嘌呤脱氨酶的支架上。在产生具有高度混杂活性的酶变体后，突变的数量将被系统地减少，以确定那些对新活性至关重要的氨基酸交换，并确定从GUAD到AtzB的合理进化轨迹。此外，最活跃的变体将与它们的非天然底物结晶，酶-配体结构将被确定，以阐明新建立的底物特异性的机制基础。以类似的方式，将分析AtzA从腺嘌呤或胞嘧啶脱氨酶的进化起源。预期的结果将提供对支持进化过程的机制和限制的洞察，特别是关于最近获得的新的酶功能。

Enzymes are powerful biocatalysts that accelerate metabolic reactions with enormous efficiency and specificity. Although it is interesting and important to understand how new enzymes have naturally evolved from existing ones, this process has barely been analyzed up to now. Obviously, the underlying mechanisms can be best investigated for enzymes that are able to degrade anthropogenic substances that have been introduced into the environment only during the last decades. Consequently, the evolution of those enzymes must have occurred within this short time frame. A well-suited example is provided by the proteins AtzA, AtzB and AtzC, which decompose the herbicide atrazine and enable their host organisms to use the reaction products as carbon and nitrogen sources. Similarities with respect to amino acid sequence, three-dimensional structure, and reaction mechanism suggest that the Atz enzymes have evolved by gene duplication and subsequent specialization from different members of the amidohydrolase superfamily subtype III, many of which deaminate nucleobases. In accordance with this hypothesis, the AtzB enzyme shows low promiscuous guanine deaminase (GuaD) activity. Within the proposed project, this activity shall be boosted by rational protein engineering based on the detailed comparison of residue conservation patterns determining the different substrate specificities of AtzB and GuaD. In the inverse direction high AtzB activity shall be established on the scaffold of the most closely related guanine deaminases. After generation of enzyme variants with highly promiscuous activities, the number of mutations will be systematically reduced to identify those amino acid exchanges that are crucial for the new activities and to determine a plausible evolutionary trajectory from GuaD to AtzB. Moreover, the most active variants will be crystallized with their non-native substrates and the enzyme-ligand structures will be determined to elucidate the mechanistic basis of the newly established substrate specificities. In a similar manner, the evolutionary origin of AtzA from an adenine or a cytosine deaminase will be analyzed. The expected results will provide insights into mechanisms and constraints that underpin evolutionary processes, particularly with respect to the very recent acquisition of new enzymatic functions.