Experimental reconstruction of the molecular evolution of atrazine-degrading enzymes

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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.