Thermo-L-Asparaginases: From the Role in the Viability of Thermophiles and Hyperthermophiles at High Temperatures to a Molecular Understanding of Their Thermoactivity and Thermostability

L-asparaginase (L-ASNase) is a vital enzyme with a broad range of applications in medicine, food industry, and diagnostics. Among various organisms expressing L-ASNases, thermophiles and hyperthermophiles produce enzymes with superior performances—stable and heat resistant thermo-ASNases. This review is an attempt to take a broader view on the thermo-ASNases. Here we discuss the position of thermo-ASNases in the large family of L-ASNases, their role in the heat-tolerance cellular system of thermophiles and hyperthermophiles, and molecular aspects of their thermoactivity and thermostability. Different types of thermo-ASNases exhibit specific L-asparaginase activity and additional secondary activities. All products of these enzymatic reactions are associated with diverse metabolic pathways and are important for mitigating heat stress. Thermo-ASNases are quite distinct from typical mesophilic L-ASNases based on structural properties, kinetic and activity profiles. Here we attempt to summarize the current understanding of the molecular mechanisms of thermo-ASNases’ thermoactivity and thermostability, from amino acid composition to structural–functional relationships. Research of these enzymes has fundamental and biotechnological significance. Thermo-ASNases and their improved variants, cloned and expressed in mesophilic hosts, can form a large pool of enzymes with valuable characteristics for biotechnological application.


Introduction
Thermophiles and hyperthermophiles are unique microorganisms that not only survive but also thrive at high temperatures. Among environmental factors temperature is unique in that it crosses physical barriers [1]. As a result, organisms cannot efficiently shield themselves from temperature in the way they can shield themselves from extreme external pH or salinity by maintaining steep concentration gradients over biological membranes. Each biomolecule inside microorganisms must be adapted to the temperatures in which they grow [1].
Thermophiles and hyperthermophiles are generally categorized based on the optimum growth temperatures. No consensus has been reached regarding the exact temperature range of each category [1][2][3]; in this review we use the following: thermophiles (TP) grow in the range of temperatures from 50 • C to 80 • C, whereas hyperthermophiles (HP) live in extremely hot environments with temperatures over 80 • C.
To thrive in such habitats, microorganisms use a combination of adaptive mechanisms. Studies of these specific mechanisms are carried out on each level of hierarchy, from microbial communities to cells and molecules. The area of special interest is elucidation of the molecular basis of thermophilic proteins' stability and functionality under extreme conditions. Intrinsic resistance of proteins to high temperatures remains a fascinating Most of the identified enzymes from thermophilic bacteria represent bacterial-type II L-ASNases (Table 1). Examples include enzymes from Streptomyces thermoluteus subsp. fuscus NBRC 14270 (GenBank accession no. BAJ25701.1) [46], Melioribacter roseus (GenBank accession no. WP_014855710.1) [49], Thermus thermophilus (GenBank accession no. BDB12174.1), Thermus aquaticus (GenBank accession no. WP_003047338). Although being defined as type II L-ASNases, poor sequence identity between some of these enzymes is  At the same time, the sequence identity between hyperthermophilic bacterial-type I L-ASNases from archaea and type I L-ASNase from mesophilic bacteria Escherichia coli (EcAI, GenBank accession no. NP_416281.1, encoded by the ansA gene) used as a representative of this type [30,38] does not exceed 37% (Figure 1).
Although characterization of thermo-ASNases is far from complete, large variations in their properties are already evident, this being in keeping with the poor sequence identity between L-ASNases of TP/HP and mesophilic origin, as well as within the thermo-ASNase group.
In general, an increasing need for a revision of L-ASNases classification is noted in recent reviews [38,51]. While Schultz da Silva et al. have proposed to rename bacterialtype L-asparaginases, plant-type L-asparaginases, and Rhizobial-type L-asparaginases as Class 1, Class 2 and Class 3, respectively [51], Lubkowski and Wlodawer are the first who suggested separating HP archaeal L-ASNases in a distinct subgroup [38]. The authors proposed to group L-ASNases into three categories: tetrameric enzymes of type I and type II, and dimeric archaeal enzymes (HP L-ASNases) [38].
Unlike tetrameric mesophilic enzymes, L-ASNases expressed in HP archaea are homodimeric and sufficiently divergent. The enzymes display structural and topological properties that are distinct from either typical type I or type II L-ASNases, have active sites that deviate from the canonical arrangement seen in type I or II ASNases, or display kinetic/activity profiles that are at the intersection between type I and II ASNases [38].
Agreeing with the opinion of Lubkowski and Wlodawer, it should be noted that only the first characterized archaeal L-ASNases-Pyrococcus horikoshii (PhA), Pyrococcus furiosus (PfA), and Thermococcus kodakarensis (TkA)-were taken into consideration [38]. According to recent data, TP/HP microorganisms can possess several enzymes of different types exhibiting L-asparaginase activity [43,49]. For example, hyperthermophilic archaeon T. kodakarensis contains not only TkA more similar to members of the type I [14], but also a newly discovered TK2246 classified as a plant-type L-ASNase [43]. TK2246 displays low similarity with both TkA and typical plant-type L-ASNase E. coli EcAIII. In the pioneering research, homology modelling revealed structural changes between TK2246 and EcAIII-the presence of additional loop in TK2246 compared to EcAIII [43]. Moreover, gel filtration chromatography and SDS-PAGE indicated that TK2246 exists in a homodimeric form comprising two identical subunits [43]. At the same time, known mesophilic plant-type L-ASNases, including EcAIII, are heterotetramers or dimers of two αβ heterodimers [30,34]. In this view, it is likely that the subgroup of archaeal L-ASNases should be further divided into additional types.
The proposed classification of Lubkowski and Wlodawer concerns only the consideration of thermo-ASNases from archaea as a separate subgroup; nevertheless, thermo-ASNases of bacterial origin are also quite distinct from typical mesophilic L-ASNases. Also, there is no structural evidence, but it is supposed, that bacterial thermo-ASNases can act in oligomeric state different from di-or tetrameric, for example hexameric L-ASNase Thermus thermophilus (trimer of dimers) [30,47].
Initial classification criteria, such as comparison of amino acid homologies to EcAI and EcAII and/or substrate affinities (millimolar vs. micromolar, expressed in terms of K M ) are also hardly applicable to thermophilic L-ASNases. For example, micromolar affinity to L-asparagine is defined as a specific trait of bacterial type II L-ASNases. Nevertheless, known type II L-ASNases from thermophilic bacteria show K M values in the millimolar range [6,49]. The level of substrate affinity in thermo-ASNases predetermined not by type, but rather the fact of adaptation of these organisms to high temperatures, far exceeding the appropriate for mesophiles. Increased K M values of various enzymes originating from thermophilic microorganisms compared to mesophilic counterparts confirm this conclusion: phosphoglycerate kinase [52], glutamate dehydrogenase [53], alkaline phosphatase [54][55][56], GTPase (TrmE) [57] and glucose-6-phosphate dehydrogenase [58]. In fact, successful attempts to improve the thermal stability of mesophilic L-ASNases have also resulted in a concomitant increase in K M [59]. It appears that adaptation at high environmental temperatures involves an increase in K M and k kat for thermo-ASNases [19]. This characteristic feature of thermo-ASNases allows optimizing catalytic efficiency by reaching a balance between substrate binding and the rate of product release.
In summary, there is a clear need to revise the current classification of L-ASNases. Considering the striking differences between mesophilic and TP/HP enzymes, probably associated with differences in the living environments, it must be recognized that thermo-ASNases must form a unique and novel group in the large family of L-ASNases and must be appropriately subdivided inside the group. This may be an extension of the classification proposed by Schultz da Silva et al., where thermo-ASNases would be classified into Class 4. Based on similarity in tertiary structures and general mechanisms within previously accepted types, and in an attempt to distinguish thermo-ASNases from mesophilic ones, Class 4 can be further subdivided into type I-like, type II-like, and plant-type-like ( Figure 2 mental temperatures involves an increase in KM and kkat for thermo-ASNases [19]. This characteristic feature of thermo-ASNases allows optimizing catalytic efficiency by reaching a balance between substrate binding and the rate of product release. In summary, there is a clear need to revise the current classification of L-ASNases. Considering the striking differences between mesophilic and TP/HP enzymes, probably associated with differences in the living environments, it must be recognized that thermo-ASNases must form a unique and novel group in the large family of L-ASNases and must be appropriately subdivided inside the group. This may be an extension of the classification proposed by Schultz da Silva et al., where thermo-ASNases would be classified into Class 4. Based on similarity in tertiary structures and general mechanisms within previously accepted types, and in an attempt to distinguish thermo-ASNases from mesophilic ones, Class 4 can be further subdivided into type I-like, type II-like, and plant-type-like ( Figure 2).

L-ASNases as Key Metabolic Enzymes for Thermophilic and Hyperthermophilic Microorganisms
From the viewpoint of a living organism, L-ASNases catalyze a specific biochemical reaction that affects a number of metabolic processes, particularly those necessary for survival of TP/HP microorganisms at high temperatures ( Figure 3).

L-ASNases as Key Metabolic Enzymes for Thermophilic and Hyperthermophilic Microorganisms
From the viewpoint of a living organism, L-ASNases catalyze a specific biochemical reaction that affects a number of metabolic processes, particularly those necessary for survival of TP/HP microorganisms at high temperatures ( Figure 3). turation caused by high-temperature stress. Due to the NH2 groups in the molecule, lysine functions as an ion-coating on the surface of membrane components and proteins to prevent denaturation. Furthermore, lysine is a kosmotropic compound due to the ammonium group in the side chain. The kosmotropic property of lysine may contribute to the protection of cells from the disordering of cellular macromolecules caused by high temperature, similar to the protective effect of compatible solutes against chaotropic stress [64]. As shown in a recent study, the increased level of lysine conferred high-temperature stress tolerance to E. coli cells [64]. Lei et al. have shown that Asp is able to enhance heat stress tolerance in plants by up-regulation of a total of nine amino acids-lysine, threonine, glutamate, asparagine, arginine, leucine, valine, glycine, and tryptophan [60]. and hyperthermophilic (HP) microorganisms under heat stress. Asp-derived-aspartate-derived, N-nitrogen, C-carbon, TCA-tricarboxylic acid cycle. (a) Aspartic acid, formed from asparagine by the action of thermo-ASNases, is associated with multiple metabolic pathways. (b) The role of thermo-ASNases in heat stress tolerance associated with the conversion of glutamine to glutamic acid: intensification of respiration and metabolism of amino acids. (c) Isoaspartyl aminopeptidase activity of thermo-ASNases as part of the protein repair system under heat-stress. (d) Maintenance of intracellular pH mediated by thermo-ASNases: thermo-ASNases by releasing ammonium ions can neutralize the excess proton influx under high temperatures. The diamond borders represent type I-like, type II-like and plant type-like thermo-ASNases. The red border is type I-like and type II-like thermo-ASNases associated with functions (a,b,d). The green border is plant type-like thermo-ASNases associated with functions (a,c,d).
Beyond its role as an amino acid in proteins, a precursor of amino acids and regulator of amino acid biosynthesis, aspartate is required for conversion of IMP to AMP in de novo L-ASNases catalyze the conversion of L-asparagine (L-Asn) to L-aspartic acid (or aspartate (Asp)) and ammonia. Asp acts as a critical metabolic hub to interconnect with diverse metabolic pathways ( Figure 3a). According to recent data, Asp provides positive effects on mitigating abiotic stresses [60].
First of all, L-ASNases play a vital role in the biosynthesis of aspartate-derived amino acids, namely lysine, threonine, methionine and isoleucine, since aspartic acid is the precursor of these amino acids ( Figure 3a) [61][62][63].
According to recent data, one of these amino acid-lysine-is involved in adaptation and tolerance to environmental stresses in various organisms [64,65]. Lysine is a charged amino acid; thus, its accumulation may contribute to the prevention of the protein denaturation caused by high-temperature stress. Due to the NH 2 groups in the molecule, lysine functions as an ion-coating on the surface of membrane components and proteins to prevent denaturation. Furthermore, lysine is a kosmotropic compound due to the ammonium group in the side chain. The kosmotropic property of lysine may contribute to the protection of cells from the disordering of cellular macromolecules caused by high temperature, similar to the protective effect of compatible solutes against chaotropic stress [64]. As shown in a recent study, the increased level of lysine conferred high-temperature stress tolerance to E. coli cells [64]. Lei et al. have shown that Asp is able to enhance heat stress tolerance in plants by up-regulation of a total of nine amino acids-lysine, threonine, glutamate, asparagine, arginine, leucine, valine, glycine, and tryptophan [60].
Beyond its role as an amino acid in proteins, a precursor of amino acids and regulator of amino acid biosynthesis, aspartate is required for conversion of IMP to AMP in de novo purine synthesis and provides the carbon backbone for de novo pyrimidine synthesis ( Figure 3a) [66]. The increased content of uracil, UMP, guanosine, and thymine by Asp under heat stress was reported for plants [60]. The authors conclude that Asp may contribute to the maintenance of RNA and DNA synthesis, supporting growth and defense against heat stress [60].
Thus, aspartate deficiency will impair protein, purine nucleotide, and pyrimidine nucleotide synthesis, resulting in decreased cell proliferation. Indeed, Sullivan et al. revealed that exogenous aspartate addition is sufficient to restore proliferation of cells that otherwise stop proliferating or die when activity of electron transport chain is impaired [66].
When excess amounts beyond the normal protein synthesis requirements are available, L-asparagine is hydrolyzed and utilized as a source of carbon and nitrogen [67]. L-ASNases of different types participate in nitrogen and carbon metabolism [35,39]. While expression of bacterial type II L-ASNases involved in carbon metabolism is tightly regulated by different factors [39], bacterial type I L-ASNases, engaged in nitrogen metabolism, appear to be expressed constitutively [30,39]. Asparagine is an important nitrogen storage and transport molecule, due to its relatively high nitrogen-to-carbon ratio (2:4, compared with 2:5 for glutamine, 1:5 for glutamic acid and 1:4 for aspartic acid, for example) and its relative chemical inertia. L-ASNases, participating in recycling the organically bound nitrogen through the ammonification process, play a notable role in nitrogen biogeochemical cycling [68].
In addition to asparaginase activity, some TP/HP L-ASNases have at least residual glutaminolytic activity [6]. Glutamate is known as an important component of several pathways, which links amino acid and respiration metabolism together (Figure 3b) [60]. Glutamate serves as a precursor of proline and arginine [60,69,70]. Arginine, in turn, is the biological precursor of nitric oxide (NO) and polyamines, which significantly contribute to stress tolerance [60,[71][72][73]. TP/HP microorganisms require polyamines for growth at high temperatures [74]. Disruption of the genes involved in polyamine synthesis in the hyperthermophile T. kodakarensis resulted in severe growth defects at 85 • C, and even more so at 93 • C, which could be slightly reversed at 85 • C but not at 93 • C, by supplying exogenous polyamine spermidine [74].
T. thermophiles is also known to produce a variety of polyamines; the most common ones, spermidine and spermine, are synthesised using a distinct pathway from arginine via aminopropyl agmatine [1,71]. Polyamines of T. thermophilus were found to be necessary for the maintenance of the ribosome, tRNA His , and tRNA Tyr structural integrity during growth at high temperatures [75].
In vitro biochemical studies indicate that polyamines induce structural changes to DNA that are proposed to facilitate growth at extreme temperatures [74,76].
In addition to glutaminase activity, Asp itself can increase accumulation of glutamate and arginine under heat stress. Asp-mediated activation of glutamate metabolism and the TCA enhances heat tolerance [60].
Thus, the direct metabolites of the biochemical reactions catalyzed by L-ASNases-Asp and Glu-are important nodes in the metabolic network of TP/HP microorganisms (Figure 3a,b).
Plant-type L-ASNases identified for TP/HP microorganisms [43,49] are known to have dual L-asparaginase and isoaspartyl aminopeptidase activity [35]. Isoaspartyl aminopeptidase activity protects plants from the spontaneous accumulation of highly toxic βasparagine dipeptides (isoAsp) [35]. The role of plant-type L-ASNases in mesophilic microorganisms that also possess other types of the enzyme is not clear. Obviously, that is not the case with thermophiles and hyperthermophiles. The rate of isoAsp residue formation increases 910-fold at 90 • C compared with 23 • C [77]. Accumulation of proteins with altered aspartyl-and asparaginyl-residues is believed to be detrimental to cell survival at elevated temperatures [78]; hence, modified or damaged protein (peptide) should be rapidly digested to prevent cellular damage and to provide a potential nutrient source to support cell survival in harsh environments.
According to recent experimental data, heat shock-induced protein aggregates retarded growth of thermophilic bacteria, but expression of β-aspartyl peptidase (BAP) alleviated the growth defect by degrading damaged proteins [78]. BAP can directly hydrolyze isopeptide bonds, resulting in the release of Asp, and this might be an efficient repair mechanism for handing β-aspartyl-containing peptides [78].
Protein homeostasis, a balanced state between folded proteins and protein aggregates, is critical for cellular metabolism and physiology. In extremophiles thriving under harsh environments, in which proteins are vulnerable to protein inactivation and aggregation, cellular protein repair systems play a pivotal role in protein quality control to support cellular integrity and survival [78]. Spontaneous isopeptide bond formation, in particular, isoAsp residues, is accelerated at elevated temperatures [77,79], thus TP/HP L-ASNases with isoaspartyl aminopeptidase activity (plant-type L-ASNases) seems to act as a part of cellular protein repair system important for survival under extremely high temperatures (Figure 3c).
On the other hand, these enzymes may utilize β-aspartyl peptides as a source of amino acids to provide protein biosynthesis under stress conditions, for example, under nutrient depletion.
Another aspect of L-ASNase activity is releasing ammonium ions. Ammonia represents a direct metabolite of the biochemical reaction induced by the enzyme, and exerts profound effects on the electrochemical gradient, membrane potential, and the intracellular pH, thereby affecting the synthesis of ATP and activity of membrane transporters (Figure 3d) [80][81][82]. Singh et al. were the first who reported the influence of L-ASNase on intracellular pH-regulation and H + -gradient through ammonia releasing [81].
Ammonia (NH 4+ ), the product of the enzymatic hydrolysis of Asn/Gln, could be involved in counteracting the H + -influx (acid stress) [81]. Indeed, it is known that the proton and sodium permeabilities of all biological membranes increase with the temperature [83]. The upper temperature border of life depends not only on the stability of the biomolecules, but, in addition, the proton permeability of membranes, which at higher temperatures may become too high to maintain electrochemical proton gradients in order to gain energy [84]. The increased motion of the lipid molecules in the membranes at high temperature causes an increased proton permeability, and in turn, the high proton leakage makes it impossible to control intracellular pH. Among TP/HP microorganisms, TP bacteria have more difficulties to restrict the proton permeation [83].
Thermo-ASNases by releasing ammonium ions can neutralize the excess proton influx, thus maintaining the much required intracellular environment (Figure 3d). The enzyme may be crucial in providing an immediate protective response in maintaining pH and aiding in survival at stress conditions [81]. It is interesting that E. coli exploits a similar survival mechanism to maintain an intracellular pH under environmental acidic stress: glutaminase converts Gln to glutamate (Glu) with concomitant release of ammonia, which neutralizes protons, resulting in elevated intracellular pH under acid stress [85].
Overall, these data confirm that thermo-ASNases are key metabolic enzymes, being a part of the complicated heat-tolerance cellular system in TP/HP microorganisms. All products of the enzymatic reaction are associated with multiple metabolic pathways. Playing crucial roles in the integration of cell metabolic systems, they contribute to survival under high temperature stressful conditions.

Toward a Molecular Understanding of Thermo-ASNases Thermostability
Thermo-ASNases are optimally active at temperatures close to, slightly below, or even above the optimal growth temperature of the host organisms (Table 1). An analysis of the relationships between growth temperatures and optimal catalytic temperatures of enzymes revealed the influence of speciation rather than a common feature. Thus, archaeal thermo-ASNases from Pyrococcus sp. have optimum below the growth temperature. Another example is the thermo-ASNase from A. fulgidus, an enzyme that has an optimum of activity at 70 • C and the host strain has an optimum of growth at 83 • C.
According to previous data, many individual enzymes in TP and HP have catalytic optima much lower than the growth temperatures [1]. In addition, even the average activity of enzymes isolated from these organisms is 10-20 • C lower than the growth temperature. Extrinsic factors are proposed to provide enzyme adaptation with an optimum below the growth temperature in addition to adaptations in protein sequence and fold [1]. These factors may include compatible solutes such as diglycerol phosphate, found as a mechanism of protein thermostabilization in the hyperthermophiles, in particular, in archaea A. fulgidus [86].
Another character of the relationship between growth temperatures and enzyme optima can be observed for Thermococcus sp. These archaeal thermo-ASNases display an optimum higher than the growth temperature. It is likely that these enzymes themselves can serve as part of the first-line defense system under stress conditions, including heat stress. More active under elevated temperatures, they can quickly stabilize cellular systems providing an additional margin of safety for host cells.
Thermostability and optimal activity at high temperatures are inherent properties of thermo-ASNases. The differences in thermostability between TP/HP and mesophilic L-ASNases are striking. Previous studies reported that T. zilligii L-ASNase showed a slight decrease in activity after incubation for 2 h at 70-85 • C (Table 1) [17]. The halflife of T. kodakaraensis L-ASNase and T. gammatolerans L-ASNase at 85 • C was more than 120 min [15,87]. Storage of P. yayanosii L-ASNase at 37 • C for 1 month showed that 90% of the enzyme activity was retained [20]. The thermostability of non-thermophilic bacterial L-ASNases II is poor compared to thermo-ASNases. When incubated at 70 • C for 30 min, 80% of EcAII activity was lost. The half-life of EcAII at 50 • C (T (1/2, 50 • C) ) was estimated to be 60 min [59]. Mesophilic L-ASNase II from Bacillus subtilis retained approximately 14.7% of its activity after 2 h incubation at 50 • C and showed 9.0% residual activity after 2 h incubation at 60 • C [88].
Current studies agree that there is no unique feature or single mechanism responsible for the high functional activity at elevated temperatures and remarkable heat stability of TP/HP proteins [89][90][91]. A concerted action of structural, dynamic and other physicochemical attributes is utilized to ensure the delicate balance between stability and functionality of proteins at such harsh conditions.

Primary Structure Analysis
All proteins, independent of their origin, consist of the 20 canonical amino acids; thus, amino acid composition and the interaction between them in the native TP/HP enzyme structures contribute in their unique characteristics. Correlations between protein thermostability and amino acid composition include a decrease in content of chemically labile residues, a decrease in chain flexibility, an increase in average hydrophobicity, and an increase in frequency of aromatic residues [89][90][91].
The comparison of residue contents in HP/TP and mesophilic L-ASNases revealed more charged residues-Glu, Lys-in the sequences of TP/HP origin (Figure 4a,b). Most thermo-ASNases contain fewer uncharged polar residues (Asn, Gln, His, Cys) and increased residue hydrophobicity (Ile, Val). The same preferential usage of amino acids in thermal adaptation was investigated by comparative proteome analysis, using genomes from mesophiles, thermophiles, and hyperthermophiles ( Figure 4)   It is worth mentioning that elevated temperatures trigger not only cysteine oxidation, but also such spontaneous chemical modifications as deamidation. Two deamidation mechanisms are known for Asn and Gln residues [91]. Thus, as part of the adaptation mechanism against deamidation at high temperatures, some TP/HP enzymes contain less Asn and Gln [91,94]. These amino acids are also less common in thermo-ASNases ( Figure  4a).
In summary, the results of the primary structure analysis of thermo-ASNases support the conclusion that thermophilic adaptation cannot be defined only in terms of significant differences in the amino acid composition [91]. Probably, the distribution of the residues, their positions and interactions can affect the thermoactivity and thermostability of TP/HP proteins to a greater extent. Camillau and Claverie have reported that thermophilic proteins have less Gln, Ala, and His on their surfaces than mesophilic proteins do and more charged residues on their surfaces, particularly Lys and Glu [93].

Structural Features and Key Residues Essential for Thermoactivity and
With some variation in the content of individual amino acids, the total abundance of charged residues, accompanied by a decrease in the content of polar/uncharged amino acids, is the major feature of thermo-ASNases (Figure 4b).
In general, comparative analysis of the amino acid composition of TP/HP and mesophilic L-ASNases shows trends with variations between and inside these groups (Figure 4, Supplementary Materials, Table S1). For example, cysteine's high sensitivity to oxidation at high temperature suggests that TP/HP enzymes contain fewer cysteines than their mesophilic counterparts [91]. Thermo-ASNases of hyperthermophilic origin on average contain lower number of cysteine residues (Figure 4a). However, the percentage of Cys residues is higher in the enzymes from thermophilic bacteria (S. thermoluteus and M. roseus) (Supplementary Materials, Table S1).
Initially, it was believed that susceptibility of cysteines and disulfide bridges to destruction at high temperatures determined the upper limit for the stability of proteins containing disulfide bridges [91]. Nevertheless, recent observations indicate, cysteines that are present in proteins from hyperthermophiles are often involved in specific stabilizing interactions (e.g., disulfide bridges and metal liganding) and/or are inaccessible to the solvent [91]. Disulfide bridges can serve as a stabilization strategy, and conformational environment and solvent accessibility are determining factors in the protection of disulfide bridges against destruction. Thermo-ASNase from hyperthermophilic archaea P. furiosus PfA that grows at optimum temperature of 100 • C confirms the fact that not all disulfide bridges have equal susceptibility to destruction. PfA contains two cysteines (Supplementary Materials, Table S1) and displays stability in harsh conditions, including denaturation at 8 M concentration of urea [24].
It is worth mentioning that elevated temperatures trigger not only cysteine oxidation, but also such spontaneous chemical modifications as deamidation. Two deamidation mechanisms are known for Asn and Gln residues [91]. Thus, as part of the adaptation mechanism against deamidation at high temperatures, some TP/HP enzymes contain less Asn and Gln [91,94]. These amino acids are also less common in thermo-ASNases (Figure 4a).
In summary, the results of the primary structure analysis of thermo-ASNases support the conclusion that thermophilic adaptation cannot be defined only in terms of significant differences in the amino acid composition [91]. Probably, the distribution of the residues, their positions and interactions can affect the thermoactivity and thermostability of TP/HP proteins to a greater extent.

Structural Features and Key Residues Essential for Thermoactivity and Thermostability of Thermo-ASNases
Elucidation of the available L-ASNase crystal structures from hyperthermophilic archaea P. horikoshii, P. furiosus, and T. kodakaraensis in comparison with the enzymes from mesophiles revealed increased content of salt bridges in thermo-ASNases-17.5 ÷ 20.0% vs. 9.2 ÷ 13.9% for the non-thermophilic enzymes [14]. While no significant differences were observed between these groups in the number of hydrogen bonds in helix structures t [14].
According to previous studies, most thermophilic proteins tend to have more salt bridges than their mesophilic homologs [95][96][97].
Chan et al. demonstrated that stabilizing salt bridges enhance the thermostability of proteins by reducing the heat capacity change in unfolding ∆Cp. In particular, the double-mutant-cycle approach revealed that breaking a salt bridge increased the ∆Cp value, and conversely, the formation of each salt-bridge contributed to a reduction in ∆Cp by 0.8-1.0 kJ mol −1 K −1 . A decrease in ∆Cp due to the extra salt bridges in thermophilic proteins, in turn, enhances protein thermostability by up-shifting and broadening the protein stability curve so that the protein remains stable at a wider range of temperatures [97].
An in-depth evaluation of the role of distinct inter-residue interactions and individual amino acid contributions for maintaining thermoactivity and thermostability of thermo-ASNases was carried out by Li et al. [59]. The rationale for the difference in thermostability of the thermophilic and non-thermophilic L-ASNases was based on the comparative analysis of the representative enzymes from P. yayanosii CH1 (PyA), T. gammatolerans (TgA), B. subtilis (BsAII), and E. coli (EcAII).

Identification of Amino Acid Residues Essential for Thermoactivity and Thermostability of Thermo-ASNases
Despite significant divergence in amino acid sequences of L-ASNases, the architecture and composition of their active-site pockets are highly conserved [38,59,98]. So-called "special residues" are located near to the highly conserved regions in thermo-ASNaseshighly conserved amino acid residues different from that of non-thermophilic L-ASNases.
Analysis of PyA "special residue" mutants with respect to their shift in optimum temperature, activity, and the values of half-life revealed two of the nine substitutions analyzed that affect the thermostability and optimum temperature of the enzyme-D51G and K298L (Figure 5a,c). The half-lives at 85 • C (T (1/2, 85 • C) ) of mutants D51G and K298L were 70 min and 55 min lower than those of the wild type, respectively. D51G and K298L had 30 • C and 10 • C reduction in optimum temperature compared to PyA, respectively ( Table 2) [59].
Residue D51 is located on a loop around the active site. The mutation D51G reduced thermostability of the enzyme due to the deficiency of two polar contacts with the surrounding residues, which led to an increase in the surface charge near the active site [59]. Unlike residue D51, K298 was located at the core of the α-helix (residues 291-305) far away from the active site [59]. Compared with mutated residue K298L, residue K298 not only formed polar contacts with the residues (A294, T297, I302) in the α-helix (residues 291-305) but also formed contacts with C terminal residues (L315, M316, T318, and E233). The mutated residue K298L led to a decrease in C terminal connectivity and resulted in decreased thermostability. Thus, K298 represents one of key residues responsible for the thermostability of thermo-ASNase. A tight junction at the C terminal is essential for maintaining high thermal stability of the enzyme [59].    Unlike residue D51, K298 was located at the core of the α-helix (residues 291-305) far away from the active site [59]. Compared with mutated residue K298L, residue K298 not only formed polar contacts with the residues (A294, T297, I302) in the α-helix (residues 291-305) but also formed contacts with C terminal residues (L315, M316, T318, and E233). The mutated residue K298L led to a decrease in C terminal connectivity and resulted in decreased thermostability. Thus, K298 represents one of key residues responsible for the thermostability of thermo-ASNase. A tight junction at the C terminal is essential for maintaining high thermal stability of the enzyme [59].
Analysis of conformational dynamics revealed that both mutations reduced overall tightness of the protein structure that resulted in decreased thermostability of the enzyme.
The obtained results were confirmed using thermo-ASNase from archaea T. gammatolerans. Mutation of the corresponding residues D52G and K298L showed decreased T (1/2, 85 • C) ( Table 2) [59]. In addition, the mutated enzymes D52G and K298L displayed a lower optimum temperature, though to a different extent (Table 2).
Since the "special" residues D51 and K298 of PyA (corresponding to residues D52 and K298 of TgA, respectively) are highly conserved in 1000 homologous enzymes, they can be considered essential to maintaining thermoactivity and thermostability of a large number of thermo-ASNases.
These findings were confirmed in the reverse experiments using EcAII and BsAII. Corresponding to the thermo-ASNase mutations D−G and K−L, the mutated forms EcA II G57D and L305K showed a higher thermostability, with the half-life at 50 • C (T (1/2, 50 • C) ) 40 min and 30 min longer than that of EcAII, respectively (Table 2). Improved thermostability was also revealed after substitutions at the corresponding residues G107D and L354K of BsAII [59].
The results suggest that "special" residues D51 and K298 of PyA (corresponding to D52 and K298 of TgA, G57 and L305 of EcAII, G107 and L354 of BsAII) make L-ASNases adapted to different working temperatures.

Key Secondary Structures Associated with the Thermal Adaptation of Thermo-ASNases
Not only amino acid residues affect the enzyme characteristics, but also the secondary structures. In attempts to identify the key secondary structures responsible for thermostability of the thermo-ASNase PyA, the secondary structures near the highly conserved and "special" residues-PyA-β, PyA-loop, PyA-α1, PyA-α2, PyA-α3, and PyA-α4-were replaced with the corresponding structure of the mesophilic EcAII [59].
While PyA-loop, PyA-α1, PyA-α2, and PyA-α4 were lethal and PyA-β showed no significant influence on activity and stability under high temperatures, the mutation PYA-α3 (Figure 5e) showed a lower optimum temperature and shorter T (1/2, 85 • C) compared to the wild-type enzyme (Table 3). Based on the protein structure model, replacing of α3 resulted in the cleavage of the helical structure and a decrease in three polar contacts with the surrounding amino acid residues, resulting in the formation of a more flexible structure. Moreover, the mutant had more positive surface charge and increased the root mean square fluctuation (RMSF) [59]. These results indicated that flexibility of the structure coupled with high positive surface charge decreased thermal stability of PyA thermo-ASNase after structural mutation. However, two additional polar contacts with the surrounding residues appeared in the mutated structure α3 of TgA [59]. Together with a reduced positive surface charge, these changes resulted in improved thermostability compared to the wild-type enzyme (Table 3).
Thus, it was revealed, that α3 ( Figure 5e) exerted a great impact on the thermostability of thermo-ASNases, though the effect can be different depending on the character of interaction with the surrounding amino acid residues.

The Role of C-Terminal Amino Acid Residues in Thermo-ASNase Thermostability
Leng et al. emphasized that thermophilic proteins tend to insert or delete residues in the C-terminal domain [99]. The C-terminal domain has been shown to play a vital role in the stabilization of the structure and is proposed to contribute to the thermostability of thermophilic proteins [99,100].
Thermo-ASNases have extended C-terminal region with additional 8-9 amino acid residues compared to mesophilic L-ASNases II. Considering that the C-terminal residues often display a significant impact on the level of protein thermostability, truncated forms of thermo-ASNases were obtained and analyzed [59]. Li et al. have found that the Cterminal residues affected thermostability of thermo-ASNase from P. yayanosii CH1 (PyA), but not from T. gammatolerans [59]. PyA-∆C9 (Figure 5b) displayed a 45 min shorter half-life (T (1/2, 85 • C) ) with minimal change in optimal temperature.
Analysis of the mutants PyA-∆1-PyA-∆8 revealed that the 6th C-terminal residue of PyA-E323-affects the thermostability of thermo-ASNase. Truncation of 6 C-terminal residues reduced T (1/2, 85 • C) from 105 min for the wild type to 60 min for the mutant [59]. E323 is probably a key residue for the thermal stability of PyA, which is involved in multiple interactions with C-terminal loop and core residues. The loss of connection between the C-terminal and the helix (residues 292-305) after mutation led to protein instability [59].
Thus, the C-terminal residues were identified as a structural determinant of PyA thermo-ASNase thermal stability. The cPfA displayed improved specific activity and similar thermophilic profile compared to the wild type full-length protein PfA. At 80 • C the specific activity was increased from 338 U/mg for PfAwt to 413 U/mg for cPfA [102]. However, further cutting of the terminal short β-strand in the N-terminal domain revealed that this structure is important in providing stability under high temperature conditions. For the resulting defunct form dcPfA (lacking both linker and LVVN peptide), an increase in temperature triggered the formation of high molecular weight aggregates-assemblies with non-specific oligomerization sites-leading to a decrease in stability of the protein [101]. The most significant decrease in dcPfA activity was observed at temperatures above 50 • C. The loss of activity for dcPfA was 38%, 60%, 80% and 90% compared to cPfA, at 45 • C, 60 • C, 70 • C and 80 • C, respectively.
Thus, short β-strand residues L179-V-V-N182 of the N-terminal domain affect thermostability of PfA Supplementation of the peptide LVVN to the defunct enzyme restored structural framework with mesophile-type functionality.

Contribution of the Oligomeric State to the Thermostability of Thermo-ASNases
Oligomerization is recognized as a mechanism for protein stabilization to confer a thermophilic adaptation [103]. In general, proteins isolated from thermal environments are oligomers that have large hydrophobic cores and increased electrostatic interactions, disulfide bonds, salt bridging, and surface charges [104].
Since L-ASNases are active only in the oligomeric form, oligomerization is a key process in formation of the active site structure of this enzyme and its substrate specificity. Most mesophilic L-ASNases, including EcA, EwA, L-ASNases of Helicobacter pylori, Pectobacterium carotovorum, exist as homotetramers with a molecular mass of 140-150 kDa [6]. In contrast to mesophilic counterparts, thermo-ASNases, in particular, known hyperthermophilic L-ASNases, are supposed to exist in dimeric form (at ambient temperatures) [6].
In the crystallographic study of thermo-ASNase from hyperthermophilic archaea T. kodakaraensis KOD1-TkA-it was revealed that each subunit of the enzyme consists of an N-terminal and a C-terminal α/β domain connected by a short linker region [14]. The larger N-terminal domain is formed by eight β-sheet folds (β1, β4-8, β11, β12) and four α-helices (α1-4). It has been shown that the β-hairpin composed of strands β2 and β3 is highly flexible. This β-hairpin is involved in substrate binding and catalysis, and adopts "open" or "closed" conformation by TkA subunits [14].
The substrate specificity and activity of L-ASNases are directly related to the oligomerization process. TkA is active only in the dimeric form (in low temperature studies) [14]. Substrate recognition and catalytic activity involve both subunits of the enzyme, particularly, Thr11, Tyr21, Ser54, Thr55, Thr85, Asp86 and Lys156, as well as Tyr233 and Glu275, which are highly conserved for L-ASNases [14].

Folding, Association, and Intersubunit Interactions as Part of Thermo-ASNase Thermostability
Particular interest in the oligomerization of L-ASNases from microorganisms living at elevated temperatures is associated not only with the fact that oligomerization determines the enzyme activity (active sites are located in the contact zone of subunits), but also with the fact that for proteins with high hydrophobicity at elevated temperatures the formation of aggregates during assembly is highly probable; in this context, the oligomerization mechanism can be an evolutionary adaptation, which helps to avoid accumulation of these aggregates [22,23].
Using thermo-ASNase from archaea P. furiosus PfA as a model system representing homodimeric (at ambient temperatures) L-ASNase of hyperthermophilic origin, quaternary structure formation has been studied [22,23]. It has been shown that the more stable N-terminal domain plays a decisive role in the assembly of PfA. The process operates sequentially: the part of the polypeptide chain that forms the N-terminal domain folds and forms a stable core, after which the folding of the C-terminal domain occurs. For PfA asparaginase, folding and association of domains are coupled, which leads to high kinetic stability and prevents aggregation [22,23].
The N-domain of PfA acts like a folding scaffold and assists the folding of remaining polypeptide [23]. The coupled process of folding and oligomerization apparently represents the evolutionary adaptation that avoids accumulation of partially folded monomeric intermediates with a high probability of aggregation at elevated temperatures.
Thermal stability and high activity at elevated temperatures are explained by a strong intersubunit interaction. The structural analysis of the PfA enzyme showed that the monomers are assembled in a head-to-tail fashion, where the N-terminal domain of the monomer (NPfAI) is linked to the C-terminal domain (CPfAII) [23]. The two C-domains, CPfAI and CPfAII, were found to align centrally. Analysis revealed that for individual domains the intra-monomeric H-bonding and hydrophobic interactions are more as compared to intermonomeric domain-wise interactions. When all the inter-monomeric interactions are combined (NPfAI-CPfAII, CPfAI-CPFAII and NPfAII-CPfAI), they far exceeded the intra-monomeric interactions. The numbers of salt bridges in the contact zone between the monomers were also found to exceed their number inside the NPfA-CPfA monomer [23]. Overall data suggest that the extreme thermal stability of PfA is due to the presence of high intersubunit associative forces supported by extensive H-bonding and ionic interactions network.
Analyzing thermostable proteins, Reed et al. highlighted tighter packing of the hydrophobic core of TP proteins due to favorable hydrophobic interactions and increased number of ionic interactions as one of the most prominent factors of thermal adaptation to retain structure and function under extreme temperatures [104].

Heat-Induced Thermo-ASNase Super-Stable Assembly
The reduction in the oligomeric state can abolish the high thermostability of enzymes [105]. As rule, a higher oligomeric state is favorable for TP proteins [104]. In this view, it is interesting that the biological assembly of both type I and II L-ASNases of mesophilic origin is a homotetramer, whereas most of the known hyperthermophilic L-ASNases are supposed to exist in dimeric form [6]. The results of Sharma et al. shed some light on the situation: most of the structural information on thermo-ASNase was obtained from low temperature studies; in fact, at elevated temperatures, the formation of higher order oligomeric states of thermo-ASNase occurs [101].
As relevant shape-function information cannot be extrapolated from partial low temperature data, Sharma et al. have performed the high temperature small-angle X-ray scattering (SAXS) experiments along with enzyme assay to obtain new information on the structural organization of thermo-ASNase from P. furiosus PfA at higher temperatures [101]. Analysis revealed that PfA remains predominantly dimer (about 70 kDa) from 25 to 50 • C. Upon further increase in temperature, the molecular masses of particles in solution were clearly bigger than dimer, and at 80 • C the estimated mass was closer to that of tetramer of PfA. The experiments indicated that though the dimer existed at ambient temperature, heating induced higher order association.
Based on the available crystal structures and interaction analysis, 4 hydrogen bonds, 2 salt bridges and 44 hydrophobic interactions between domains were revealed for PfA tetramer [101]. Most of the residues involved in interactions belong to the terminal region of the N-terminal domain depicting that this region might be involved in molecular associations.

The Possible Role of Substrate Binding on the Oligomerization Properties of Thermo-ASNases
Substrate binding can also play a role in oligomerization and stabilization of thermo-ASNases at elevated temperatures.
The hypothesis of substrate-induced oligomerization switch was first proposed for thermo-ASNases by Sharma et al. [101]. Analysis of the crystal structures of the enzyme from P. furiosus PfA and its form cPfA, lacking a 19-residue long linker which connects N-, C-domains (Figure 5d), revealed, that the unstructured active-site loop in ligand-free enzyme became structured once the active site was occupied by the ligand [102]. The structured active sites of adjoining protein molecules established contact through H-bonds and hydrophobic interactions [101]. The number of interactions between protein molecules was approximately double, indicating that they were capable of forming more stable assemblies in the presence of substrate (Table 4) [101]. Thus, the authors concluded that although thermo-ASNase PfA existed in an oligomeric state under high temperature conditions, it is more likely that conformational changes in the presence of substrate significantly enhanced its tendency to form stronger interactions and higher order associations [101]. The influence of substrate binding on the oligomerization properties of different proteins was reported previously [106][107][108]. As an example, it was revealed that substrate stabilizes dihydrodipicolinate synthase by locking the conformation of the tight dimer unit, thus promoting docking of the tight dimers together to yield the more active tetrameric form [106]. According to the crystal structure analysis, substrate binding promoted greater contacts at the dimerization interface, which essentially reduced the conformational flexibility of the dimer; that, in turn, promoted tetramerization [106]. Moreover, it was associated with an increase in the stability of the enzyme. In the thermal denaturation experiments the apparent melting temperatures increased from 63 • C in substrate-free form to 74 • C in the presence of the substrate. An increased thermal stability was confirmed to be specific to the substrate, but no effect was observed in the presence of substrate analogues [106].
Lactate dehydrogenase was also reported to undergo an obvious conformational change upon substrate binding and stabilized against thermal denaturation by its substrates, NADH and lactate [109].
In summary, until recently nothing was known about the ability of substrate binding to stabilize thermo-ASNases at elevated temperatures. A novel mechanism for ligand-induced stabilization revealed for the enzyme from hyperthermophilic archaea P. furiosus offers insight into the synergy between quaternary structure, protein dynamics, and catalytic function for adaptation to hot environments.

Conclusions
The research interest in L-ASNases is primarily due to their wide application in biotechnology. In the search for forms with improved efficacy-enhanced activity and stability-many microbial species, from archaeal, bacterial and eukaryotic sources, have been screened for L-ASNase production. Among them, thermophiles and hyperthermophiles produce L-ASNases with extraordinary properties-stable and heat resistant thermo-ASNases with superior performances for biotechnology.
In writing this review we have attempted to present a broader view of the thermo-ASNases: from the types currently identified, to highlighting striking differences from mesophilic counterparts, proposal for their classification, summary of their physiological role as a part of the complicated heat-tolerance cellular system in TP/HP microorganisms, and interpretation their thermostability and optimal activity at high temperatures at the molecular scale.
Nature created ingenious molecular mechanisms for maintaining thermoactivity and thermostability of thermo-ASNases, so that they protect their host from extreme heat.
Obviously, the fact that TP/HP usually possess a set of L-asparaginases in the genomes, and these enzymes are expressed at a high level, is not surprising in view of the predicted role of thermo-ASNases in the integration of cell metabolic systems for survival under high temperatures.
Getting insight into the mechanisms used by thermo-ASNases to modulate their thermoresistance is a way to better understand how TP/HP adapt to these extreme environments. The results obtained so far confirm that no single mechanism is responsible for the remarkable stability of thermo-ASNases. All levels of adaptation-from amino acid composition to structural features, protein dynamics, and catalytic function-provide synergy. The findings summarized in the review may pave the way to rationally engineer both thermo-ASNases suitable for special application at moderate temperatures and their mesophilic counterparts with enhanced stability. This can revolutionize their future-oriented applications.