CONFORMATIONAL DYNAMICS OF ANCESTRAL THID-HMPPK ENZYMES FROM MESOPHILIC AND THERMOPHILIC BACTERIAL LINEAGES

Thermophilic proteins remain stable at temperatures above 60 °C due to adaptations that allow them to function efficiently under these conditions. One commonly described adaptation is reduced conformational dynamics at room temperature compared to their mesophilic homologs. However, the features that confer thermostability are not universal, as evolutionary history can influence the adaptive strategies employed. Moreover, the correlation between increased thermostability and decreased conformational dynamics in thermophilic proteins has recently been questioned.

ThiD-HMPPK enzymes are ATP-dependent phosphotransferases involved in key steps of the bacterial vitamin B1 biosynthesis pathway. In this work, we compared the conformational dynamics of two ancestral ThiD-HMPPK enzymes: one from a mesophilic lineage (Enterobacteriales, ancEn, Tₘ = 59 °C) and another from a thermophilic lineage (Thermus, ancTh, Tₘ = 87 °C), using small-angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations.

Kratky analysis of the SAXS data revealed similar global flexibility for both proteins at room temperature. However, ancTh displayed a trend toward reduced flexibility as the temperature approached 50 °C. Gaussian accelerated MD (GaMD) simulations showed overall enhanced conformational dynamics for the thermophilic protein ancTh compared to ancEn at both 300 K and 330 K. Interestingly, certain regions of ancEn, particularly the N- and C-termini, displayed higher fluctuations. Enzymatic assays revealed that ancTh exhibited 40-fold lower activity than ancEn at room temperature, but comparable activity at 60 °C. To assess this issue, conventional MD simulations were performed, for the enzyme-ligand ternary complexes, revealing greater flexibility in ancTh at 300 K, which decreased at 330 K.

Overall, our results suggest that the thermophilic ancestral enzyme ancTh exhibits increased conformational dynamics at room temperature relative to its mesophilic counterpart, ancEn, and point to a potential link between conformational dynamics and catalytic efficiency in these ancestral enzymes

This work was supported by the Chilean National Fund for Scientific and Technological Development (FONDECYT) through the following grants: FONDECYT Regular 1221667, FONDEQUIP EQM200202, and FONDEQUIP EQM200266.