Crystal structure of a reconstructed ancestor of Triosephosphate isomerase from eukaryotes

Abstract

Function, structure, and stability are strongly coupled in obligated oligomers, such as triosephosphate isomerase (TIM). However, little is known about how this coupling evolved. To address this question, five ancestral TIMs (ancTIMs) in the opisthokont lineage were inferred. The encoded proteins were purified and characterized, and spectroscopic and hydrodynamic analysis indicated that all are folded dimers. The catalytic efficiency of ancTIMs is very high and all dissociate into inactive and partially unfolded monomers. The placement of catalytic residues in the three-dimensional structure, as well as the enthalpy-driven binding signature of the oldest ancestor (TIM63) resemble extant TIMs. Although TIM63 dimers dissociate more readily than do extant TIMs, calorimetric data show that the free ancestral subunits are folded to a greater extent than their extant counterparts are, suggesting that full catalytic proficiency was established in the dimer before the stability of the isolated monomer eroded. Notably, the low association energy in ancTIMs is compensated for by a high activation barrier, and by a significant shift in the dimer-monomer equilibrium induced by ligand binding. Our results indicate that before the animal and fungi lineages diverged, TIM was an obligated oligomer with substrate binding properties and catalytic efficiency that resemble that of extant TIMs. Therefore, TIM function and association have been strongly coupled at least for the last third of biological evolution on earth. DATABASES: PDB Entry: 6NEE. ENZYMES: Triosephosphate isomerase 5.3.1.1, Glycerol-3-phosphate dehydrogenase 1.1.1.8.