Abstract
AbstractThe 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-interface into AncCDT-5 (a monomeric reconstructed ancestor of PaCDT). We find that single interface mutations can switch the oligomeric state of the variants (implying evolutionarily metastable oligomeric states) and that trimerization corresponds with a reduction in the KM value of the enzyme from a promiscuous level to the physiologically relevant range. We show that this can be rationalized at the structural and dynamic level by reduced sampling of a non-catalytic conformational substate, and that trimerization was likely followed by a C-terminal extension that further refined the conformational sampling and kinetic properties of the enzyme. This work provides insight into how neutral sampling of metastable oligomeric states along an evolutionary trajectory can facilitate the evolution and optimization of enzyme function.Importance & Impact StatementUnderstanding how and why structural complexity (including homo-oligomerization and sequence insertions) emerges during the evolution and diversification of natural enzymes is a key goal in the study and design of protein function. We show that cyclic homo-oligomeric states can emerge via a small number of substitutions, and that trimerization and a C-terminal extension contributed to the tuning of catalytic properties during the evolution of cyclohexadienyl dehydratase from Pseudomonas aeruginosa.
Publisher
Cold Spring Harbor Laboratory