Enhancing the Catalytic Activity of Thermo-Asparaginase from by a Double Mesophilic-like Mutation in the Substrate-Binding Region

Overview

Journal Int J Mol Sci

Publisher MDPI

Specialties Biochemistry
Chemistry
Molecular Biology

Date 2023 Jun 10

PMID 37298582

Authors

Maria Dumina

Dmitry Zhdanov

Alexander Zhgun

Marina Pokrovskaya

Svetlana Aleksandrova

Alexander Veselovsky

Michael Eldarov

Affiliations

Soon will be listed here.

Abstract

L-asparaginases (L-ASNases) of microbial origin are the mainstay of blood cancer treatment. Numerous attempts have been performed for genetic improvement of the main properties of these enzymes. The substrate-binding Ser residue is highly conserved in L-ASNases regardless of their origin or type. However, the residues adjacent to the substrate-binding Ser differ between mesophilic and thermophilic L-ASNases. Based on our suggestion that the triad, including substrate-binding Ser, either GSQ for meso-ASNase or DST for thermo-ASNase, is tuned for efficient substrate binding, we constructed a double mutant of thermophilic L-ASNase from (TsA) with a mesophilic-like GSQ combination. In this study, the conjoint substitution of two residues adjacent to the substrate-binding Ser55 resulted in a significant increase in the activity of the double mutant, reaching 240% of the wild-type enzyme activity at the optimum temperature of 90 °C. The mesophilic-like GSQ combination in the rigid structure of the thermophilic L-ASNase appears to be more efficient in balancing substrate binding and conformational flexibility of the enzyme. Along with increased activity, the TsA D54G/T56Q double mutant exhibited enhanced cytotoxic activity against cancer cell lines with IC values from 2.8- to 7.4-fold lower than that of the wild-type enzyme.

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