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

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

Journal

International Journal of Molecular Sciences
ISSN: 1661-6596 , E-ISSN: 1422-0067

Output data

Year: 2025, Volume: 26, Number: 12, Article number : 5437, Pages count : DOI: 10.3390/ijms26125437

Authors

Dumina Maria V. ¹ , Zhdanov Dmitry D. ² , Veselovsky Alexander V. ² , Pokrovskaya Marina V. ² , Aleksandrova Svetlana S. ² , Minyaev Mikhail E. ³ , Varfolomeeva Larisa A. ¹ , Matyuta Ilya O. ^1,4 , Boyko Konstantin M. ¹ , Zhgun Alexander A. ¹

Affiliations

1	Federal Research Center “Fundamentals of Biotechnology” of the Russian Academy of Sciences, Moscow 117312, Russia
2	Institute of Biomedical Chemistry, Moscow 119121, Russia
3	N.D. Zelinsky Institute of Organic Chemistry Russian Academy of Sciences, Moscow 119071, Russia
4	Moscow Center for Advanced Studies, Moscow 123592, Russia

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 TsAID54G/T56Q mutant, which exhibited increased activity after a double mutation in the substrate-binding region. TsAI and TsAID54G/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 TsAID54G/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.

Dumina M.V. , Zhdanov D.D. , Veselovsky A.V. , Pokrovskaya M.V. , Aleksandrova S.S. , Minyaev M.E. , Varfolomeeva L.A. , Matyuta I.O. , Boyko K.M. , Zhgun A.A.
Hyperthermophilic L-Asparaginase from Thermococcus sibiricus and Its Double Mutant with Increased Activity: Insights into Substrate Specificity and Structure
International Journal of Molecular Sciences. 2025. V.26. N12. 5437 . DOI: 10.3390/ijms26125437 WOS Scopus OpenAlex

≡ Web of science:	WOS:001516228100001
≡ Scopus:	2-s2.0-105009022261
≡ OpenAlex:	W4411097400