Asparaginase (ASNase) is a biopharmaceutical for Acute Lymphoblastic Leukemia (ALL) treatment. However, it shows undesirable side effects such as short lifetimes, susceptibility to proteases, and immunogenicity. Here, ASNase encapsidation was genetically directed in bacteriophage P22-based virus-like particles (VLPs) (ASNase-P22 nanoreactors) as a strategy to overcome these challenges. ASNase-P22 was composed of 58.4 ± 7.9% of coat protein and 41.6 ± 8.1% of tetrameric ASNase. Km and Kcat values of ASNase-P22 were 15- and 2-fold higher than those obtained for the free enzyme, respectively. Resulting Kcat/Km value was 2.19 × 10⁵ M⁻¹ s⁻¹. ASNase-P22 showed an aggregation of 60% of the volume sample when incubated at 37 °C for 12 days. In comparison, commercial asparaginase was completely aggregated under the same conditions. ASNase-P22 was stable for up to 24 h at 37 °C, independent of the presence of human blood serum (HBS) or whether ASNase-P22 nanoreactors were uncoated or PEGylated. Finally, we found that ASNase-P22 caused cytotoxicity in the leukemic cell line MOLT-4 in a concentration dependent manner. To our knowledge, this is the first work where ASNase is encapsulated inside of VLPs, as a promising alternative to fight ALL.

Asparaginase (ASNase) is a widely applied chemotherapeutic drug that is used to treat Acute Lymphoblastic Leukemia (ALL); however, immune responses and silent inactivation of the drug often limit its bioavailability. Many strategies have been proposed to overcome these drawbacks, including the development of improved formulations (biobetters), but only two of them are currently on the market. Nano- and micro-encapsulation are some of the most promising and novel approaches to enhance in vivo performance of ASNase, preventing the direct contact of the enzyme with the environment, protecting it from protease degradation, increasing the enzymes catalytic half-life, and in some cases, reducing immunogenicity. This review summarizes the strategies, particularly for ASNase nano- and micro-encapsulation, and their main findings, constraints, and current gaps in the state-of-the-art knowledge. The pros and cons of the use of different nanocarriers are discussed with the idea to ultimately provide safer and more effective treatments for patients with ALL.

In plants, the last step in the biosynthesis of the osmoprotectant glycine betaine (GB) is the NAD⁺-dependent oxidation of betaine aldehyde (BAL) catalyzed by some ALDH10 enzymes that exhibit betaine aldehyde dehydrogenase (BADH) activity. Given the irreversibility of the reaction, the short-term regulation of these enzymes is of great physiological relevance to avoid adverse NAD⁺/NADH ratio decreases. We here report that the Spinacia oleracea betaine aldehyde dehydrogenase ( So BADH) is reversibly and partially inactivated by BAL in the absence of NAD⁺ in a time- and concentration-dependent mode. Crystallographic evidence indicates that the non-essential Cys⁴⁵⁰ ( So BADH numbering) forms a thiohemiacetal with BAL, totally blocking the productive binding of the aldehyde. Interestingly, in contrast with Cys⁴⁵⁰ the catalytic cysteine (Cys²⁹¹) did not react with BAL in the absence of NAD⁺. The trimethylammonium group of BAL binds in the same position in the inactivating or productive modes. Accordingly, BAL does not inactivate the C450S So BADH mutant and the degree of inactivation of the A441I and A441C mutants corresponds to their very different ability to bind the trimethylammonium group. Cys⁴⁵⁰ and the neighboring residues that participate in stabilization of the thiohemiacetal are strictly conserved in plant ALDH10 enzymes with proven or predicted BADH activity, suggesting that inactivation by BAL is a common feature of them. Under osmotic stress conditions, this novel partial and reversible covalent regulatory mechanism may contribute to prevent NAD⁺ exhaustion, while still permitting the synthesis of high amounts of GB and avoiding the accumulation of the toxic BAL.

Polymorphism is frequently observed from different crystallization conditions. In proteins, the effect on conformational variability is poorly documented, with only a few reported examples. Here, three polymorphic crystal structures determined for a large-subunit catalase, CAT-3 from Neurospora crassa, are reported. Two of them belonged to new space groups, P1 and P4₃2₁2, and a third structure belonged to the same space group, P2₁2₁2₁, as the previously deposited 2.3 Å resolution structure (PDB entry 3ej6 ), but had a higher resolution (1.95 Å). Comparisons between these polymorphic structures highlight the conformational stability of tetrameric CAT-3 and reveal a distortion in the tetrameric structure that has not previously been described.