Many cancers utilize L-glutamine as a major energy source. This “L-glutamine addiction” involves a well-characterized pathway whereby L-glutamine is hydrolyzed by a glutaminase (GLS) to L-glutamate, which is then converted to α-ketoglutarate, the carbons of which enter the tricarboxylic acid (TCA) cycle. However, mammalian tissues/cancers possess a rarely mentioned alternative pathway (the glutaminase II pathway): L-Glutamine is transaminated to α-ketoglutaramate (KGM), followed by ω-amidase (ωA)-catalyzed hydrolysis of KGM to α-ketoglutarate. Uncertainty may prevail over the name glutaminase II which may be confused with the enzyme named glutaminase 2 (GLS2). Thus, we recently suggested a new name for the glutaminase II pathway, namely the glutamine transaminase-ω-amidase (GTωA) pathway. Herein, we 1) evaluate three recent articles that mention L-glutamine addiction, but not the GTωA pathway, 2) summarize the metabolic importance of the GTωA pathway, including its role in closing the methionine salvage pathway, and 3) as a source of anaplerotic α-ketoglutarate. An advantage of the GTωA pathway [i.e., L-glutamine + α-keto acid + H2O α-ketoglutarate + L-amino acid + +NH4] is that it is irreversible and that there is no net change in redox status, permitting α-ketoglutarate production during hypoxia. Finally, we discuss possible clinical benefits of GTωA pathway inhibitors.

L-asparaginase from bacterial sources have been used in antineoplastic treatments and the food industry. A novel type II L-a sparaginase encoded by the N-truncated gene ansZP21 of halotolerant Bacillus subtilis CH11 isolated from Chilca salterns in Peru was expressed using a heterologous system in Escherichia coli BL21 (DE3)pLysS. The recombinant protein was purified using one-step nickel affinity chromatography and exhibited activity of 234.38 U mg-1 and a maximum catalytic activity at pH 9.0 and 60 °C. The enzyme shows a homotetrameric form with an estimated molecular weight of 155 kDa by gel filtration chromatography. The enzyme half-life at 60 °C was 3 h 48 min, and L-asparaginase retained 50% of initial activity for 24 h at 37 °C. The activity was considerably enhanced by KCl, CaCl2, MgCl2, mercaptoethanol, and DL-dithiothreitol (p-value < 0.01). Moreover, the Vmax and Km were 145.2 µmol mL-1 min-1 and 4.752 mM, respectively. These findings evidence a promising novel type II L-asparaginase for future industrial applications.

Acute lymphoblastic leukaemia is currently treated with bacterial L-Asparaginase; however, its side effects bring up the special need for improved and efficient novel enzymes development. Previously, we obtained low anti-asparaginase antibody production and high serum enzyme half-life in mice with the P40S/S206C mutant; however, its specific activity was significantly reduced. Thus, our aim was to test single mutants, S206C and P40S, for in vitro and in vivo assays. Our results showed that the drop in specific activity was caused by P40S substitution. Besides, our single mutants were highly stable in biological environment simulation, unlike double mutant P40S/S206C. The in vitro cell viability assay demonstrated that mutant enzymes have higher cytotoxic effect than WT on T-cells derived ALL and on some solid cancer cell lines. The in vivo assays were performed in mice to identify toxicological effects, to evoke immunological response and to study the enzymes pharmacokinetics. From these tests, none of the enzymes was toxic; however, S206C evoked lower physiological changes and immune responses. In relation to pharmacokinetic profile, S206C has two-fold higher activity than WT and P40S two hours after injection. In conclusion, we present bioengineered E. coli asparaginases with high specific enzyme activity and lesser side effects.

Acute lymphoblastic leukemia (ALL) is the most common cancer among children worldwide, characterized by an overproduction of undifferentiated lymphoblasts in the bone marrow. The treatment of choice for this disease is the enzyme L-asparaginase (ASNase) from bacterial sources. ASNase hydrolyzes circulating L-asparagine in plasma, leading to starvation of leukemic cells. The ASNase formulations of E. coli and E. chrysanthemi present notorious adverse effects, especially the immunogenicity they generate, which undermines both their effectiveness as drugs and patient safety. In this study, we developed a humanized chimeric enzyme from E. coli L-asparaginase, which would reduce the immunological problems associated with current L-asparaginase therapy. For these, the immunogenic epitopes of E. coli L-asparaginase (PDB: 3ECA) were determined and replaced with those of the less immunogenic Homo sapiens asparaginase (PDB:4O0H). The structures were modeled using the Pymol software and the chimeric enzyme was modeled using the SWISS MODEL service. A humanized chimeric enzyme with four subunits similar to the template structure was obtained, and the presence of asparaginase enzymatic activity was predicted by protein-ligand docking.

Cervical cancer is caused by a persistent and high-grade infection, caused by the Human Papilloma Virus (HPV), which, when entering cervical cells, alters their physiology and generates serious lesions. HPV 18 - is among those most involved in carcinogenesis in this region, but there are still no drug treatments that cause cure or total remission of lesions caused by HPV. Knowing that L-Asparaginase is an amidohydrolase, which plays a significant role in the pharmaceutical industry, particularly in the treatment of specific cancers, due to its antitumor properties, some studies have demonstrated its cytotoxic effect against cervical cancer cells. However, the commercial version of this enzyme has side effects, such as hypersensitivity, allergic reactions and silent inactivation due to the formation of antibodies. To mitigate these adverse effects, several alternatives have been explored, including the use of L-asparaginase from other microbiological sources, which is the case with the use of the fungus Aspergillus niger, a high producer of L-asparaginase. The study investigated the influence of the type of fermentation, precipitant, purification, characterization and in vitro cytotoxicity of L-asparaginase. The results revealed that semisolid fermentation produced higher enzymatic activity and protein concentration of A. niger. The characterized enzyme showed excellent stability at pH 9.0, temperature of 50ºC, resistance to surfactants and metallic ions, and an increase in enzymatic activity was also noted with the organic solvent ethanol. Furthermore, it exhibited low cytotoxicity in GM and RAW cells, and significant cytotoxicity in HeLa cells. These findings indicate that L-asparaginase derived from Aspergillus niger may be a promising alternative for pharmaceutical production. Its attributes, including stability, activity and low toxicity in healthy cells, suggest that this modified enzyme could overcome challenges associated with antitumor therapy.

L-asparaginase (ASNase) is an aminohydrolase currently used in the pharmaceutical and food industries. Enzyme immobilization is an exciting option for both applications, allowing a more straightforward recovery and increased stability. High surface area and customizable porosity make carbon xerogels (CXs) promising materials for ASNase immobilization. This work describes the influence of contact time, pH, and ASNase concentration on the immobilization yield (IY) and relative recovered activity (RRA) using Central Composite Design methodology. The most promising results were obtained using CX with an average pore size of 4 nm (CX-4), reaching IY and RRA of 100%. At the optimal conditions, the ASNase-CXs biocomposite was characterized and evaluated in terms of kinetic properties and operational, thermal and pH stabilities. The immobilized ASNase onto CX-4 retained 71% of its original activity after six continuous reaction cycles, showed a good thermal stability at 37 °C (RRA of 91% after 90 min) and was able to adapt to both acidic and alkaline environments. Finally, the results indicated a 3.9-fold increase in the immobilized ASNase affinity for the substrate, confirming the potential of CXs as a support for ASNase and as a cost-effective tool for subsequent use in the therapeutic and food sectors.

The effect of high temperature (HT) stress on nicotine biosynthesis in Nicotiana attenuata was examined. Nicotine content was measured in mature leaves, young sink leaves and in roots from well-watered plants grown at 25 °C as controls and from plants exposed to 38 °C and 43 °C temperatures applied for 24, 48, 72 and 96-hr duration. At 38 °C, all leaf nicotine levels were significantly less than control plants for up to 72-hr exposure but rose sharply thereafter to levels significantly greater than controls with 96-hr exposure. In contrast, plants exposed to 43 °C never exhibited a reduction in leaf nicotine content and showed a significant increase in content with just 48-hr exposure. When radioactive 11CO2 was administered to plants, HT stress significantly reduced fixation. Furthermore, radiocarbon flux analysis revealed that ‘new’ C partitioning (as 11C) into the [11C]-amino acid pool was significantly reduced with HT stress as were yields of [11C]-aspartic acid, an important amino acid in nicotine biosynthesis, and its beta-amido counterpart [11C]-asparagine significantly reduced. In contrast, [12C]-aspartic acid levels appeared unaffected at 38 °C but were significantly elevated at 43 °C relative to controls. Additionally, [12C]-asparagine levels were significantly elevated at both stress temperatures. Finally, leaf total soluble protein (TSP) was reduced 39% with long exposures to both stress temperatures. However, Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) which was 41% TSP appeared unaffected by HT stress. Altogether, these results support the theory that plant proteins other than Rubisco degrade at elevated temperatures freeing up essential amino acids supporting the nicotine biosynthetic machinery, and while feedback might downregulate de novo amino acid synthesis from ‘new’ C resources, more than likely plant C/N rebalancing caused by HT reductions in C input restricted N uptake, which in turn, limited this process.