Temperature is a critical environmental factor that profoundly influences the survival and functionality of organisms. Over evolutionary timescales, organisms adapt to different thermal conditions by fine-tuning the properties of their enzymes. Rather than restructuring the enzyme architecture outright, temperature adaptation is primarily achieved through specific amino acid substitutions that modulate enzyme dynamics and stability [1,2]. These sequence-level changes allow the enzyme to maintain an optimal balance between flexibility and rigidity, ensuring proper function across temperature extremes. Proteins that remain active in high-temperature environments often contain amino acid substitutions that reduce flexibility — for example, by introducing proline residues into loop regions—to enhance structural rigidity and resist thermal denaturation. In contrast, cold-adapted enzymes frequently feature substitutions that increase local flexibility, such as the incorporation of glycine residues in loops, allowing them to retain catalytic efficiency under conditions of reduced thermal motion [[3], [4], [5], [6]]. Through these finely tuned residue-level modifications, homologous enzymes are able to maintain both stability and activity in diverse temperature ranges.

In this study, we investigated the temperature sensitivity of enzymatic activity through molecular modeling and structural analysis of mesophilic ketosteroid isomerase (KSI) from Pseudomonas putida, which exhibits optimal catalytic performance at 303 K [7,8]. KSI catalyzes the isomerization of Δ5-3-ketosteroid to Δ4-3-ketosteroid via intramolecular transfer of the C4β proton to the C6β position [9,10]. In the studied KSI, Y16, D40, and protonated D103 are the three catalytically essential residues. Y16 and D103 form hydrogen bonds with the substrate's C3 carbonyl oxygen, thereby stabilizing substrate binding—particularly the oxyanion in the transition state—and helping to orient the steroid core for catalysis. This arrangement enables D40 to approach the β-face of the substrate and function as a general base, abstracting the C4β proton and subsequently donating it to the C6β position, thus completing the isomerization (Fig. S1) [11,12].

Another key feature of KSI is its requirement for dimerization to achieve catalytic activity, as the monomeric form is inactive [6,13]. In this study, we further investigated the structural consequences of dimerization by comparing the monomeric and dimeric states of KSI through molecular dynamics simulations. This analysis allowed us to identify conformational differences induced by dimer formation, shedding light on how dimerization contributes to maintaining the structural integrity necessary for enzymatic function.

For decades, computational approaches—particularly molecular dynamics (MD) simulations—have served as powerful tools for monitoring protein dynamics and structural details, providing insights into how structural fluctuations relate to biological function. These methods have been instrumental in bridging the gap between static structures and the dynamic behaviors critical for enzymatic activity [[14], [15], [16], [17], [18], [19], [20]]. While experimental techniques such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy have significantly advanced our understanding of protein structures [[21], [22], [23]], they primarily capture proteins in static conformational states, offering limited information on their inherent flexibility and dynamic transitions. In the present study, we initiated our analysis using the high-resolution X-ray crystallography structure of KSI (PDB: 6C17). Building upon this static framework, we extended our investigation to include both monomeric and dimeric forms under optimal and elevated temperature conditions—303 K and 338 K, respectively. Through this molecular modeling study, we aimed to address two fundamental questions: how elevated temperature impacts the structural integrity of KSI, and how dimerization converts an inactive catalytic site into an active one.