Structural and biophysical aspects of l-asparaginases: a growing family with amazing diversity

Abstract

l-Asparaginases 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-asparaginases, focusing on the mechanistic aspects of the well established types and speculating about the potential of the new members of this amazingly diversified family.

Keywords: active site; amidohydrolases; catalytic mechanism; l-asparaginases; leukemia; nucleophiles.

Figure 1

Overview of selected events in the history of l-asparaginases in science and medicine. Scientific discoveries intertwine with medical applications of l-­asparaginases. References to the historical facts presented are included in the text.

Figure 2

Structure-based classification of l-asparaginases. Historically, l-asparaginases were named according to the source organism of the first enzymes that were discovered. The new nomenclature divides l-asparaginases into three classes. Cytosolic type 1 enzymes are expressed constitutively, while the expression of type 2 enzymes, which are secreted to the periplasm, is induced under anaerobic conditions in E. coli. In the R. etli-type Class 3, constitutive type 4 enzymes are thermostable, while type 5 enzymes are considered thermolabile and their expression is induced by the presence of l-Asn. Examples of enzymes are listed below the boxes. The organism name abbreviations are as follows: Ec, Esherichia coli; Pf, Pyrococcus furiosus; Ph, Pyrococcus horikoshii; Tk, Thermococcus kodakarensis; Vc, Vibrio cholerae; Yp, Yersinia pestis; Cp, Cavia porcellus; Er, Erwinia chrysanthemi; Ew, Erwinia carotovora; Cj, Campylobacter jejuni; Hp, Helicobacter pylori; Ws, Wolinella succinogenes; Ll, Lupinus luteus; Pv, Phaseolus vulgaris; Hs, Homo sapiens; Re, Rhizobium etli. Examples of PDB codes are summarized in Supplementary Table S1. An asterisk denotes enzymes that are annotated as products of an ansA gene; however, the architecture of the active site and substrate affinity suggest classification as Class 1 type 2 enzymes.

Figure 3

Architecture of bacterial type 2 asparaginases. (a) Protomer of EcAII (PDB entry 3eca) with the N-terminal domain in pink, the C-terminal domain in orange, the linker in red, the hinge region (HR) in blue and the flexible gating element (FGE) in green. (b) The EcAII homotetramer (a dimer of two intimate dimers) has D ₂ (222) symmetry with intimate dimers A/B (pink/yellow) and C/D (green/blue). (c) Detailed view of the EcAII active site with l-­Asp bound. (d) Location of the active site in the A/B dimer.

Figure 4

(a) Conformational changes accompanying the allosteric regulation of EcAI: left, without l-Asn (PDB entry 2p2d); right, in the presence of l-Asn (PDB entry 2p2n). (b) l-Asn located in the active site and allosteric site of the EcAI protomer.

Figure 5

(a)–(e) Steps in the catalytic mechanism of type 2 bacterial (Class 1) l-asparaginases based on the reactions proposed by Lubkowski et al. (2020 ▸). Residues and waters (3×) involved in proton shuttling from ⁽¹⁾Thr to ⁽⁴⁾Asp via ⁽²⁾Tyr are colored blue. ⁽³⁾Thr and ⁽⁵⁾Lys that exchange protons with conserved water W2 (red) are colored green. ⁽⁴⁾Ala (carbonyl O atom) and W1 creating the oxyanion hole are colored magenta (the carbonyl O atom of ⁽¹⁾Thr, which is also part of the oxyanion hole, is not shown). ⁽⁶⁾Ser participating in substrate/product anchoring, the l-Asn substrate, intermediate products and the l-Asp end product are colored black (substrate/product is also hydrogen-bonded to ⁽⁷⁾Gln, ⁽⁸⁾Ala, ⁽⁹⁾Asn′ and ⁽¹⁰⁾Glu′, but these interactions are not shown in the figure). (f)–(j) Steps of the catalytic mechanism of Class 2 l-asparaginases based on the reactions proposed by Nomme et al. (2012 ▸). Protein residues involved in catalysis are colored blue, residues creating the oxyanion hole are colored magenta and water molecules important for catalysis are colored red, ⁽¹⁰⁴⁾Arg important for substrate anchoring is colored black (substrate/product is also hydrogen-bonded to ⁽¹⁰³⁾Thr, ⁽¹⁰⁶⁾Gly, ⁽¹⁰⁵⁾Asp and ⁽¹⁰⁷⁾Gly, but these interactions are not shown in the figure). In all panels, the directions of the nucleophilic attacks are shown by brown arrows. For clarity, only the functional groups of the protein residues are shown in atomic detail; the remainder is marked by a circle. In all panels, the substrate/product molecules are colored black and shaded.

Figure 6

Details of the catalytic mechanism of bacterial (Class 1) type 2 asparaginases. The panels show structures of the K162M mutant of EcAII, in which ⁽⁵⁾Lys was replaced by ⁽⁵⁾Met* . (a) Acyl-enzyme intermediate (PDB entry 6v2g). (b) Tetrahedral intermediate TI2 (PDB entry 6v25). Residues and waters (W1, W2, W3) participating in proton shuttling from ⁽¹⁾Thr to ⁽⁴⁾Asp via ⁽²⁾Tyr are colored cyan. ⁽³⁾Thr and ⁽⁵⁾Met* (corresponding to ⁽⁵⁾Lys in the wild-type protein) are colored green. Red dashed lines represent hydrogen bonds between waters and residues involved in catalysis. Magenta dotted lines show the interactions stabilizing the TI2 intermediate in the oxyanion hole formed by the carbonyl O atom of ⁽⁸⁾Ala and the conserved water molecule W1. A fat pink arrow marks the position of water W2 in (a), which is incorporated into TI2 in (b). Black dashed lines represent hydrogen bonds anchoring the substrate/product in the active site. Some less important hydrogen bonds were omitted for clarity. A brown curved arrow indicates the direction of the nucleophilic attack of water W2 on the acyl-enzyme intermediate.

Figure 7

Crystal structure of the mature form of the Class 2 potassium-dependent l-asparaginase from P. vulgaris (PDB entry 4pv2). The mature protein is a heterotetramer built of two α subunits (green and magenta) and two β subunits (cyan and yellow). All Class 2 l-asparaginases possess a stabilization loop with a coordinated Na⁺ ion (blue sphere). Potassium-dependent enzymes also possess an activation loop that is capable of coordinating a K⁺ ion (brown sphere).

Figure 8

(a)–(c) Schematic representation of the mechanism of maturation of Class 2 asparaginases. The process is started by the nucleophilic attack of ⁽¹⁰¹⁾Thr on the scissile bond ⁽¹⁰⁰⁾Gly- ⁽¹⁰¹⁾Thr (black). The reaction needs a water molecule (red) and assistance of the general base B. The oxyanion hole (orange) is made by ^(Na)Asn from the stabilization loop and another water molecule (orange). At the end of the maturation process, the protomer is cleaved into two subunits: α (green) and β (blue).

Figure 9

Details of l-Asp (orange) binding in Class 2 asparaginases. (a) l-Asp bound in the active site of immature HsAIII (PDB entry 4pvr); a fragment of the linker region is shown in dark pink. (b) l-­Asp in the active site of mature ECAIII and the residues stabilizing its binding (PDB entry 2zal).