Structural, thermodynamic and catalytic characterization of an ancestral triosephosphate isomerase reveal early evolutionary coupling between monomer association and function

• Published in: The FEBS journal Vol. 286; no. 5; pp. 882 - 900
• Main Authors: Schulte‐Sasse, Mariana, Pardo‐Ávila, Fátima, Pulido‐Mayoral, Nancy O., Vázquez‐Lobo, Alejandra, Costas, Miguel, García‐Hernández, Enrique, Rodríguez‐Romero, Adela, Fernández‐Velasco, Daniel Alejandro
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01-03-2019
• Subjects: ancestral sequence reconstruction; Animals; Binding; Biocatalysis; Biological Evolution; Catalysis; Coding; Coupling; Crystallography, X-Ray; diffusion‐limited catalysis; Dimers; Enthalpy; enzyme evolution; Fungi; Fungi - enzymology; Glycerol; Monomers; Oligomers; Protein Binding; Protein Conformation; Protein Stability; Proteins; Spectrum Analysis - methods; Structural analysis; Structural stability; Structure-function relationships; Substrates; Thermodynamics; TIM barrel; Triose-phosphate isomerase; Triose-Phosphate Isomerase - chemistry; Triose-Phosphate Isomerase - metabolism; TIM barrel; enzyme evolution; ancestral sequence reconstruction; diffusion-limited catalysis
• DOI: 10.1111/febs.14741
• EISSN: 1742-4658
• ISSN: 1742-464X

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.
The ubiquitous glycolytic enzyme triosephosphate isomerase (TIM) is a homo‐oligomer in which structure, catalysis and stability are coupled, but how and when this mechanism evolved is poorly understood. Here, Fernández‐Velasco and colleagues address these questions using ancestral sequence reconstruction (ASR). Biochemical characterization of ancestral TIMs identified by ASR revealed that the encoded proteins are well‐folded, active dimers that dissociate into inactive but partially folded monomers. Ligand‐binding stabilizes the folded dimers. The authors conclude that ancestral TIMs were obligated oligomers sharing similar properties to extant TIMs, suggesting that association, catalysis and stability were coupled before the animal and fungi lineages diverged.