Retracing and understanding the molecular evolution of atrazine-degrading enzymes

• Applicant: Professor Dr. Reinhard Sterner
• Subject Area: Biochemistry
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 501122718

The enzymes AtzA (atrazine chlorohydrolase) and AtzB (hydroxyatrazine ethylaminohydrolase) catalyze the first two steps of the catabolic atz pathway that degrades the xenobiotic herbicide atrazine to ammonia and carbon dioxide. Due to their recent origin, AtzA and AtzB are excellent models to study the rapid emergence of novel enzymes from evolutionary precursors, which is the major goal of this project. In the last funding period, we have shown that AtzA has evolved from a melamine deaminase progenitor. Since melamine is also a xenobiotic, our focus has shifted to the question from which precursors melamine deaminases have emerged. This problem will now be addressed by a combined in silico/in vitro/in vivo approach that includes the identification of promiscuous side activities of melamine deaminases, alanine scanning of bona fide catalytically essential residues, sequence similarity networks, and ancestral sequence reconstruction. With respect to AtzB, we have experimentally retraced its emergence from an N2,N2-dimethylguanine hydrolase precursor that has acquired high hydroxyatrazine ethylaminohydrolase activity by the introduction of only four mutations close to the active site. Molecular dynamics (MD) simulations suggest that the identified evolutionary trajectories tracing this activity change go along with a gradual population shift between two different active site conformations that allow for the binding and turnover of either N2,N2-dimethylguanine or hydroxyatrazine, respectively. These findings are in accordance with the ensemble model of enzyme evolution, which shall now be further verified by the comprehensive analysis of evolutionary intermediates and endpoints using crystal structure analysis, stopped-flow kinetic analysis, and MD simulations. We will further investigate the hypothesis that AtzB has emerged from an N2,N2-dimethylguanine hydrolase rather than from a canonical guanine deaminase, because the protein scaffold allows for a higher degree of functional promiscuity and hence evolvability. Finally, we will analyze the reaction mechanism and the physiological function of N2,N2-dimethylguanine hydrolases by also characterizing those proteins whose genes are co-localized in the same operon on the genome. For this purpose, we will attempt to confirm preliminary data suggesting that the operon-encoded proteins mediate the import and subsequent degradation of N2,N2-dimethylguanine and related substances to xanthine. Taken together, our planned investigations shall contribute to a deepened understanding of molecular enzyme evolution and functional promiscuity and elucidate the biological role of N2,N2 dimethylguanine hydrolases.

• DFG Programme: Research Grants