Abstract
L-asparaginase (L-ASNase) is a key industrial enzyme significant for cancer therapy and the food industry for reducing dietary acrylamide. The hyperthermophilic L-ASNase from Thermococcus sibiricus (TsAI) was previously shown to exhibit high activity and thermostability and is promising for biotechnology. To gain insights into structure-functional relationships of TsAI, determination of the substrate specificity, kinetic parameters, structural characterization, and molecular docking were performed. TsAI characteristics were compared with the TsAI(D54G/T56Q) mutant, which exhibited increased activity after a double mutation in the substrate-binding region. TsAI and TsAI(D54G/T56Q) were found to display high activity towards D-asparagine-62% and 21% of L-asparaginase activity, respectively-and low L-glutaminase coactivity of ~5%. Restoring the mesophilic-like triad GSQ in the mutant resulted in a two-fold increase in activity towards L-asparagine compared with TsAI. Crystal structures of TsAI forms solved at 1.9 Å resolution revealed that double mesophilic-like mutation increased the flexibility of the loop M51-L57, located in close proximity to the active site. Structural superposition and mutational analysis indicate that mobility of this loop is essential for the activity of thermo-ASNases. Molecular docking, without taking into account the temperature factor, showed that, in contrast to L-asparagine interaction, D-asparagine orientation in the TsAI and TsAI(D54G/T56Q) active sites is similar and not optimal for catalysis. Under real conditions, high temperatures can induce structural changes that reduce L-ASNase discrimination towards D-asparagine. Overall, the obtained structural and biochemical data provide a basis for a more detailed understanding of thermo-ASNase functioning and possibilities to engineer improved variants for future biotechnological application.