Experimental reconstruction of the molecular evolution of atrazine-degrading enzymes

阿特拉津降解酶分子进化的实验重建

• 批准号: 501122718
• 负责人: Professor Dr. Reinhard Sterner
• 金额: --
• 依托单位: Lehrstuhl für Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/501122718?language=en
• 关键词: Experimental; reconstruction; molecular; evolution; atrazine

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.

酶是强大的生物催化剂，以极大的效率和特异性加速代谢反应。虽然了解新酶是如何从现有酶自然进化而来的是有趣而重要的，但到目前为止，这一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中引入环境的人为物质的酶，可以最好地研究其潜在机制。因此，这些酶的进化一定是在这一短时间内发生的。一个非常合适的例子是AtzA、AtzB和AtzC蛋白质，它们分解除草剂阿特拉津并使其宿主生物体能够使用反应产物作为碳源和氮源。氨基酸序列、三维结构和反应机制的相似性表明，Atz酶是通过基因复制和随后的专业化从酰胺水解酶超家族亚型III的不同成员进化而来的，其中许多是脱氨基核碱基。根据该假设，AtzB酶显示低混杂鸟嘌呤脱氨酶（GuaD）活性。在拟定项目中，应通过合理的蛋白质工程来增强该活性，该蛋白质工程基于确定AtzB和GuaD不同底物特异性的残基保守模式的详细比较。在相反的方向上，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，将系统地减少突变的数量，以鉴定对新活性至关重要的那些氨基酸交换，并确定从GuaD到AtzB的合理进化轨迹。此外，最具活性的变体将与其非天然底物一起结晶，并确定酶-配体结构以阐明新建立的底物特异性的机制基础。以类似的方式，AtzA从腺嘌呤或胞嘧啶脱氨酶的进化起源将被分析。预期的结果将提供深入了解的机制和限制，支持进化过程，特别是关于最近收购新的酶功能。