Hyperthermophilic L-Asparaginase from Thermococcus sibiricus and Its Double Mutant with Increased Activity: Insights into Substrate Specificity and Structure

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.

Keywords: L-asparaginase; hyperthermophilic enzyme; stereoselectivity; substrate specificity; thermo-L-asparaginase structure.

Figure 1

Substrate specificity of the TsAI variants.

Figure 2

TsAI structure. (A) Functional dimer of TsAI. The small domain is colored in red, the large domain in green, and the interdomain linker in magenta. The adjacent subunit is colored in gray. (B) Residues of the active site are shown as black stick model and labeled. The residues Q54 (shown as blue sticks) and T56 selected for mutation are labeled in blue.

Figure 3

Structural superposition of TsAI (green) with L-asparaginases from hyperthermophilic archaea-TkA (A), PhA (B), PfA (C), and mesophilic bacteria EcAI (D), EcAII (E). TkA, PhA, PfA, EcAI, and EcAII are shown in orange, yellow, cyan, brown, and pink, respectively. Gatekeeper is shown as red in TsAI (residues 14–29) and blue in TkA (13–28), PhA (13–28), PfA (13–28), EcAI (17–30), and EcAII (14–33). Interdomain linker is shown as magenta in TsAI (residues 185–206) and grey in TkA (184–203), PhA (182–201), PfA (182–201), EcAI (190–211), and EcAII (190–213). Other parts of structures are shown semitransparent for clarity.

Figure 4

Comparison of TsAI structures. (A) Superposition of TsAI (green) and TsAI^D54G/T56Q (cyan). Loop M51-L57 is colored in red and magenta in TsAI and TsAI^D54G/T56Q, respectively. (B) The loop M51-L57 region in the wild-type enzyme TsAI. (C) The loop M51-L57 region in the mutant TsAI^D54G/T56Q. Polar contacts are shown as grey dotted lines. Water molecules are shown as red spheres. For clarity, only the residues described in the text are shown.

Figure 5

Designed complex of thermo-asparaginase TsAI and the mutant TsAI^D54G/T56Q with asparagine enantiomers: (A) TsAI interaction with L-asparagine; (B) TsAI interaction with D-asparagine; (C) TsAI^D54G/T56Q interaction with L-asparagine; (D) TsAI^D54G/T56Q interaction with D-asparagine. H-bonds are shown as yellow lines, salt bridge as red dashed lines, and hydrophobic interactions in grey dashed lines.