Molecular dynamics insights into dimerization-dependent catalysis and thermal adaptation of mesophilic ketosteroid...

Ketosteroid isomerase (KSI), a highly conserved enzyme in the β-ketoacyl metabolic pathway, exhibits temperature-dependent functional adaptations across species. In this study, we investigated the temperature sensitivity of mesophilic KSI from Pseudomonas putida using molecular dynamics simulations. Since KSI functions as a dimer, we simulated both monomeric and dimeric forms at its optimal catalytic temperature (303 K) and at an elevated, non-optimal temperature (338 K) to evaluate how temperature and dimerization affect activation. We focused on the dynamics of three catalytically important residues-Y16, D40, and D103-where Y16 is located on the mobile α1-helix not involved in the dimer interface, D40 lies at the edge of the dimer interface, and D103 resides at the center of the core β-sheet structure that remains static in both monomeric and dimeric states. In the monomeric form at 303 K, the Y16-D40, Y16-D103, and D40-D103 pairs exhibit broader and longer separation distances than the optimal range for catalysis. Dimerization stabilizes D40, resulting in a narrower D40-D103 separation that falls within the catalytically competent range. The relatively unchanged mobility of Y16 upon dimerization suggests that Y16 undergoes an induced-fit adjustment upon substrate binding. At 338 K, although dimerization partially corrects the D40-D103 geometry, the increased conformational flexibility of Y16 indicates a reduced likelihood of achieving the substrate-induced active-site reorganization. Together, our results demonstrate that dimerization is essential for achieving the geometric organization required for catalytic activity and that elevated temperature disrupts this coordination, rendering KSI inactive.