ABSTRACT
The emergence of oligomers is common during the evolution and diversification of protein families, yet the selective advantage of oligomerization is often cryptic or unclear. Oligomerization can involve the formation of isologous head-to-head interfaces (e.g., in symmetrical dimers) or heterologous head-to-tail interfaces (e.g., in cyclic complexes), the latter of which is less well studied and understood. In this work, we retrace the emergence of the trimeric form of cyclohexadienyl dehydratase from Pseudomonas aeruginosa (PaCDT) by introducing residues that form the PaCDT trimer-interfaces into AncCDT-5 (a monomeric reconstructed ancestor of PaCDT). We find that single interface mutations can switch the oligomeric state of the variants and that trimerization corresponds with a reduction in the KM value of the enzyme from a promiscuous level to the physiologically relevant range. In addition, we find that removal of a C-terminal extension present in PaCDT leads to a variant with reduced catalytic activity, indicating that the C-terminal region has a role in tuning enzymatic activity. We show that these observations can be rationalized at the structural and dynamic levels, with trimerization and C-terminal extension leading to reduced sampling of non-catalytic conformational substates in molecular dynamics simulations. Overall, this work provides insight into how neutral sampling of distinct oligomeric states along an evolutionary trajectory can facilitate the evolution and optimization of enzyme function.
