Abstracts Category : Other

Evolution of substrate specificity and protein-protein interactions in three enzyme superfamilies

by Maximilian Plach

Superfamilies are a classication system to combine proteins that are related through a common evolutionary origin, share similar sequences, structures, and core reaction mechanisms, but exert different functions. Today, for most superfamilies tens of thousands of sequences and hundreds of structures are known and most of the different functions of their members have been elucidated. Superfamilies thus provide a formal and biologically sensible framework to study evolutionary relationships between proteins. In the present work, the frameworks of three enzyme superfamilies were utilized to get insights into several important aspects of enzyme evolution. The rst part of this work addresses the question how enzymatic mono- and bi-functionality have evolved in the superfamily of ribose-binding ()8-barrel sugar isomerases. This superfamily contains the homologous enzymes HisA and TrpF, which catalyze similar reactions in histidine and tryptophan biosynthesis, as well as the bi-functional enzyme PriA, which catalyzes both the HisA and TrpF isomerization reactions. HisA and TrpF are ubiquitous in Archaea and Bacteria, whereas PriA is only found in certain Actinobacteria. These species have lost the dedicated TrpF enzyme and PriA is consequently part of both tryptophan and histidine biosynthesis. Much has been speculated on the evolutionary relationship of these enzymes and whether the bi-functionality of PriA is a remnant from ancient evolutionary times or a more recent development in Actinobacteria. Using ancestral sequence reconstruction it was demonstrated in this work that evolutionary ancestors of modern HisA enzymes display bi-functionality, reminiscent of PriA. A detailed enzymatic characterization of three reconstructed HisA ancestors showed that they catalyze not only the HisA but also the TrpF reaction with comparable catalytic efciencies in vitro. Metabolic complementation experiments with hisA and trpF decient Escherichia coli strains furthermore demonstrated that the bi-functional HisA ancestors could support both histidine and tryptophan biosynthesis in vivo. By a combination of sequence- and network-based in silicomethods, several modern HisA enzymes were subsequently identied that possess sequence motifs typical for bi-functional PriA enzymes. The enzymatic characterization of three such modern HisA representatives revealed that they are also bi-functional, albeit to a lesser extent, although the respective organisms possess dedicated TrpF enzymes. Thus, the ancestral bi-functionality has pertained for billions of years in HisA enzymes, without any obvious selective pressure. Consequently, a new model for the evolution of HisA, TrpF, and PriA was proposed: The bi-functionality of ancient HisA variants may have played an important role in maintaining early metabolism by supporting both histidine and tryptophan biosynthesis. After the emergence of dedicated TrpF enzymes the bi-functionality of the ancestors became expendable and diminished to the level observed in modern HisA enzymes. However, theAdvisors/Committee Members: Sterner, Reinhard (advisor).