L-Asparagines have remained an intriguing research topic since their discovery ～120 years ago, especially after their introduction in the 1960s as very efficient antileukemic drugs. In addition to bacterial asparaginases, which are still used to treat childhood leukemia, enzymes of plant and mammalian origin are now also known. They have all been structurally characterized by crystallography, in some cases at outstanding resolution. The structural data have also shed light on the mechanistic details of these deceptively simple enzymes. Yet, despite all this progress, no better therapeutic agents have been found to beat bacterial asparaginases. However, a new option might arise with the discovery of yet another type of asparaginase, those from symbiotic nitrogen-fixing Rhizobia, and with progress in the protein engineering of enzymes with desired properties. This review surveys the field of structural biology of L-Asparagines, focusing on the mechanistic aspects of the well established types and speculating about the potential of the new members of this amazingly diversified family.

Synthesis of L-Asparagine

General procedure for L-Asparaginase: The Boc-protected amino acid (1 mmol) was treated with TFA (3 mL) for 15 minutes. Afterevaporation the residue was dissolved in dioxane-water (1:1, 4 mL) and the flask wasplaced in an ice bath. tert-Butylnitrite (0.13 mL, 1.1 mmol) was added and stirring wasmaintained under nitrogen at room temperature for one hour. After pouring the reactionmixture onto celite and evaporation (50 Pa, 30 °C) into a dry free-floating powder,separation was performed utilizing a CombiFlash Rf (Teledyne ISCO) automated flashchromatography apparatus by means of AcOH-MeOH-EtOAc (1:9:90) on a normal phasesilica column affording the pure a-hydroxy carboxylic acid in the yield specified. L-Asparagine was isolated by adding water (30 mL) to thereaction mixture followed by freeze drying and HPLC purification.[1]

Structural and biophysical aspects of L-asparaginase

Although first detected in animal tissue almost 120 years ago, interest later focused on bacterial L-Asparagines with micromolar substrate affinity sufficient for successful application in the treatment of leukemia. However, even in bacteria another subtype of ;L-Asparagine is present, structurally similar but with a poor K m. The plant-specific enzymes that were discovered later are now known to have very close homologs in bacteria. While their different antigenic properties would make plant-type asparaginases excellent drug candidates, their K m (millimolar) is too high for this purpose.[2]

Structurally, bacterial-type (Class 1) and plant-type (Class 2) enzymes are completely different, but their active sites have intrinsically similar features, consisting of an abundance of threonine residues that function in a double-displacement ping-pong mechanism. Threonine as an unusual nucleophile was first indicated in the crystal structure of EcAII (Swain et al., 1993 ) and was confirmed in this role by the crystal structure of archaeal proteasome (L?we et al., 1995 ). It would be tempting to speculate why threonine is the 'nucleophile of choice' in L-Asparagines. One reason might be that the nucleophile for asparagine hydrolysis does not have to be particularly strong, but it is of advantage if it has a structural feature (such as the methyl group) that would allow stereochemical control during the course of the reaction. However, we should be cautious with such speculation because the limited information that we have about R. etli asparaginases, for example about their tentative structural homology to β-lactamases, seems to indicate that in this class the nucleophile might be the more classical serine.

The asparaginases found in R. etli, a nitrogen-fixing bacterium living in symbiosis with legumes, are a relatively new addition to the asparaginase family. The full structural and mechanistic properties of these enzymes have yet to be elucidated. However, preliminary kinetic data indicate a submillimolar K m, just one order of magnitude shy of the range required for cancer therapy. It is thus possible that with suitable structure-based engineering the R. etli-type enzymes (which also have homologs in eukaryotes) could become the long-sought alternative antileukemics. Research on L-Asparagines has a fascinating history but possibly an even more exciting future.[3]

References

[1] Luo, Shangwen; Krunic, Aleksej; Chlipala, George E.; Orjala, Jimmy[Phytochemistry Letters, 2015, vol. 13, art. no. 950, p. 47 - 52]

[2] Abaji, R., Krajinovic, M. (2019). Pharmacogenetics of asparaginase in acute lymphoblastic leukemia. Cancer Drug Resistance, 2(2), 242 - 255.

[3] Loch, J. I., Jaskolski, M. (2021). Structural and biophysical aspects of L-asparaginases: a growing family with amazing diversity. IUCrJ, 8(Pt 4), 514 - 531.