Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi

Nadeem, Muhammad; Khan, Jalaluddin A.; Al-Ghamdi, Maryam A.; Khan, Mohammad Imran; Zeyadi, Mustafa

doi:10.1590/1519-6984.244735

Cited by 6 publications

(3 citation statements)

References 49 publications

(52 reference statements)

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“…In a report suggesting that Yarrowia lipolytica L-asparaginase inhibited the proliferation and growth of MOLT-4 and RAJI cells [57], the authors claimed that they investigated type II (secreted) L-asparaginase (K m not determined), but the sequence [58] indicated that the tested enzyme was an Ntn-hydrolase. In a report about anticancer properties of the Pyrococcus abyssi L-asparaginase [59], the authors described the enzyme as "L-asparaginase I". However, the provided K m value of 2.05 mM, sequence, and structure model unambiguously indicate a class 2 Ntn-hydrolase.…”

Section: Discussionmentioning

confidence: 99%

Substrate Affinity Is Not Crucial for Therapeutic L-Asparaginases: Antileukemic Activity of Novel Bacterial Enzymes

Ściuk,

Wątor,

Staroń

et al. 2024

Molecules

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L-asparaginases are used in the treatment of acute lymphoblastic leukemia. The aim of this work was to compare the antiproliferative potential and proapoptotic properties of novel L-asparaginases from different structural classes, viz. EcAIII and KpAIII (class 2), as well as ReAIV and ReAV (class 3). The EcAII (class 1) enzyme served as a reference. The proapoptotic and antiproliferative effects were tested using four human leukemia cell models: MOLT-4, RAJI, THP-1, and HL-60. The antiproliferative assay with the MOLT-4 cell line indicated the inhibitory properties of all tested L-asparaginases. The results from the THP-1 cell models showed a similar antiproliferative effect in the presence of EcAII, EcAIII, and KpAIII. In the case of HL-60 cells, the inhibition of proliferation was observed in the presence of EcAII and KpAIII, whereas the proliferation of RAJI cells was inhibited only by EcAII. The results of the proapoptotic assays showed individual effects of the enzymes toward specific cell lines, suggesting a selective (time-dependent and dose-dependent) action of the tested L-asparaginases. We have, thus, demonstrated that novel L-asparaginases, with a lower substrate affinity than EcAII, also exhibit significant antileukemic properties in vitro, which makes them interesting new drug candidates for the treatment of hematological malignancies. For all enzymes, the kinetic parameters (Km and kcat) and thermal stability (Tm) were determined. Structural and catalytic properties of L-asparaginases from different classes are also summarized.

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Section: Discussionmentioning

confidence: 99%

Substrate Affinity Is Not Crucial for Therapeutic L-Asparaginases: Antileukemic Activity of Novel Bacterial Enzymes

Ściuk,

Wątor,

Staroń

et al. 2024

Molecules

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“…Obviously, it is difficult for enzymes of mesophilic origin to cover such a wide range of conditions required for their successful application in biotechnology, especially in the food industry. Extremophiles, in particular, thermophiles, which have been reported to produce L-ASNases with unique properties, can expand the number of biotechnologically available L-ASNases [9,[14][15][16][17]. According to experimental data, thermophilic L-ASNases (thermo-ASNases) can not only occupy a vacant niche of high-temperature food technologies but also compete with mesophilic enzymes in biomedicine [9,[15][16][17].…”

Section: Introductionmentioning

confidence: 99%

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

Думина

Zhdanov

Zhgun

et al. 2023

IJMS

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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 Thermococcus sibiricus (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 IC90 values from 2.8- to 7.4-fold lower than that of the wild-type enzyme.

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“…As the main reason for the interruption of L-asparaginase treatment is the development of hypersensitivity, caused partly by the protein's high molecular weight and bacterial origin, an enzyme that leads to fewer side effects could be found in other microbial sources such as fungi, which are eukaryotic microorganisms evolutionarily closer to human beings in comparison to bacteria [15][16][17]. Furthermore, this biopharmaceutical of great pharmaceutical and industrial importance has an increasing demand in the international market that cannot be fulfilled by natural resources, thus bulk quantities of enzymes and proteins can be produced by recombinant DNA technology [18]. The heterologous expression of L-asparaginase from some microorganisms has already been reported in the literature .…”

Section: Introductionmentioning

confidence: 99%

L-Asparaginase from Penicillium sizovae Produced by a Recombinant Komagataella phaffii Strain

et al. 2022

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L-asparaginase is an important enzyme in the pharmaceutical field used as treatment for acute lymphoblastic leukemia due to its ability to hydrolyze L-asparagine, an essential amino acid synthesized by normal cells, but not by neoplastic cells. Adverse effects of L-asparaginase formulations are associated with its glutaminase activity and bacterial origin; therefore, it is important to find new sources of L-asparaginase produced by eukaryotic microorganisms with low glutaminase activity. This work aimed to identify the L-asparaginase gene sequence from Penicillium sizovae, a filamentous fungus isolated from the Brazilian Savanna (Cerrado) soil with low glutaminase activity, and to biosynthesize higher yields of this enzyme in the yeast Komagataella phaffii. The L-asparaginase gene sequence of P. sizovae was identified by homology to L-asparaginases from species of Penicillium of the section Citrina: P. citrinum and P. steckii. Partial L-asparaginase from P. sizovae, lacking the periplasmic signaling sequence, was cloned, and expressed intracellularly with highest enzymatic activity achieved by a MUT+ clone cultured in BMM expression medium; a value 5-fold greater than that obtained by native L-asparaginase in P. sizovae cells. To the best of our knowledge, this is the first literature report of the heterologous production of an L-asparaginase from a filamentous fungus by a yeast.

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Studies on the recombinant production and anticancer activity of thermostable L- asparaginase I from Pyrococcus abyssi

Cited by 6 publications

References 49 publications

Substrate Affinity Is Not Crucial for Therapeutic L-Asparaginases: Antileukemic Activity of Novel Bacterial Enzymes

Substrate Affinity Is Not Crucial for Therapeutic L-Asparaginases: Antileukemic Activity of Novel Bacterial Enzymes

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

L-Asparaginase from Penicillium sizovae Produced by a Recombinant Komagataella phaffii Strain

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