Structural analyses of adenylate kinases from Antarctic and tropical fishes for understanding cold adaptation of enzymes

Psychrophiles are extremophilic organisms capable of thriving in cold environments. Proteins from these cold-adapted organisms can remain physiologically functional at low temperatures, but are structurally unstable even at moderate temperatures. Here, we report the crystal structure of adenylate kinase (AK) from the Antarctic fish Notothenia coriiceps, and identify the structural basis of cold adaptation by comparison with homologues from tropical fishes including Danio rerio. The structure of N. coriiceps AK (AKNc) revealed suboptimal hydrophobic packing around three Val residues in its central CORE domain, which are replaced with Ile residues in D. rerio AK (AKDr). The Val-to-Ile mutations that improve hydrophobic CORE packing in AKNc increased stability at high temperatures but decreased activity at low temperatures, suggesting that the suboptimal hydrophobic CORE packing is important for cold adaptation. Such linkage between stability and activity was also observed in AKDr. Ile-to-Val mutations that destabilized the tropical AK resulted in increased activity at low temperatures. Our results provide the structural basis of cold adaptation of a psychrophilic enzyme from a multicellular, eukaryotic organism, and highlight the similarities and differences in the structural adjustment of vertebrate and bacterial psychrophilic AKs during cold adaptation.


Results
AKs from Antarctic and tropical fishes exhibited high sequence similarities but disparate thermal stabilities. The amino acid sequence of AKNc has been aligned with those of the three homologous AKs from tropical fishes in Fig. 1. The sequences of the Antarctic and tropical AKs exhibit high-level similarity. The sequence identity between AKNc and AKDr is 92%, and AKNc shares 94% sequence identities with the other two tropical AK homologues, AKXm and AKPr. These two tropical AKs are identical except at only one position (residue 184) and, interestingly, share lower sequence identities (90%) with the tropical AKDr than with the Antarctic AKNc. Notably, the N-and C-terminal regions are the most variable in the sequence alignment of the AKs. AKNc and AKDr differ by 15 residues, more than two thirds of which are located within 30 residues from the N-and C-termini of the amino acid sequence. Only 11 residues differ between AKNc and the other two tropical AKs, and six of them are found in the N-and C-terminal regions.
The T m values of the AKs were measured by circular dichroism (CD) spectroscopy (Fig. S1). The T m of AKNc was significantly lower than those of homologues from tropical fishes (Table 1), indicating the thermal stability of AKNc is substantially reduced compared with those of the other three AKs. Among the tropical AKs, AKDr was the most thermally stable. The difference in T m between AKDr and AKNc was 11.3 °C. The T m values of AKXm and AKPr were 9.2 °C and 7.0 °C, respectively, higher than that of AKNc. In a previous study of bacterial AKs, the T m difference was only 4.3 °C between psychrophilic and mesophilic homologues 23 . These results suggest that the thermal stabilities of the fish AKs reflect the temperature preferences of their source organisms, as the thermal transition of the Antarctic AKNc occurred at a substantially lower temperature than those of its homologues from tropical fishes.

Structural analyses revealed suboptimal hydrophobic packing in the central CORE domain of AKNc.
To assess the structural basis of cold adaptation, we determined the crystal structures of AKNc and AKDr to resolutions of 1.99 and 1.75 Å, respectively, using molecular replacement. Data collection and refinement statistics are summarized in Table 2. The asymmetric units of both structures contain two independent AK monomers. The root mean square deviation (RMSD) values of the Cα atomic positions between the two monomers were only ~0.6 Å for both AKNc and AKDr, indicating that the two monomeric structures in the same asymmetric units are very similar. We hereafter describe only those (chain A's), which exhibit lower average B factors.
The chain folds of AKNc and AKDr are essentially identical to those of its homologues (Figs 2a, 3a and S2). The structures comprised the characteristic three-domain arrangement: the CORE (residues 1-38, 69-136, and 143-193), AMP bind (residues 39-68), and LID (residues 137-142) domains. The CORE domain consists of a five-stranded parallel β-sheet (β1-5) and seven α-helices (α1, α4-9), and the AMP bind domain includes two α-helices (α2, α3). The LID domain is a short loop connecting α6 and α7 helices, as is true of other AK1 structures. The co-crystallized ligand P 1 ,P 5 -di(adenosine 5′)-pentaphosphate (Ap 5 A), which mimics both AMP and ATP substrates, is bound to the active site and covered by the AMP bind and LID domains, indicating that the crystal structures of AKNc and AKDr adopt the closed conformational state of AK 28 .
To identify key structural features for the cold adaptation of AKNc, we focused on amino acid residues conserved among the three tropical AKs, but not in the Antarctic AKNc. We found three such positions (residues 28, 48, and 188) in the sequence alignment (Fig. 1a). The three AKs from the tropical fishes including AKDr have Ile28, Ala48, and Lys188 at these positions, whereas AKNc has Val28, Ser48, and Thr188. We thought that these residue substitutions might be related to the temperature adaptation of the AKs. In the structure of AKNc, Val28 in the N-terminal α1 helix interacts closely (<4 Å) with conserved hydrophobic residues in the β3 strand (Leu91) and the C-terminal α9 helix (Phe183, Val186, and Ile190) (Fig. 2b). Moreover, the Val-to-Ile mutation at this position (residue 28) found in the tropical homologues is expected to improve the hydrophobic packing in the CORE domain. In the structure of AKDr, the longer side chain of Ile28 enhances the hydrophobic contacts with the residues in β3 and α9, and interacts more closely with hydrophobic residues in β1 and β4 (Fig. 3b). In contrast, Ala48 and Lys188 are largely exposed to the solvent in the AKDr structure, and do not make close contacts with ScIeNtIfIc REPORtS | 7: 16027 | DOI:10.1038/s41598-017-16266-9 residues that are located distantly in the polypeptide (Fig. 3d,e), indicating that mutations at these two positions in AKNc may not exert significant effects on the intramolecular interactions.
In the CORE domain of AKNc, we found two additional Val residues (Val118 and Val173), around which hydrophobic packing could be improved by mutations (Fig. 2c). At these positions, AKXm and AKPr also have Val residues, but AKDr, which is the most thermally stable of the three tropical AKs, has Ile residues. Val118 and Val173 of AKNc are adjacent to each other (<4 Å) in β4 and β5, respectively. Their side chains are found in the hydrophobic interior of the CORE domain, making contacts with residues in β1, α1, and α9 such as Val13, Lys21, Val182, and Val186. However, there is room for improvement in hydrophobic packing around the two Val residues (Val118 and Val173). Val-to-Ile mutations at these positions would most likely result in closer hydrophobic contacts with residues in the β strands (β1, β4, β5) and the two terminal helices (α1, α9). In the crystal structure of AKDr, Ile118 and Ile173 improve CORE packing cooperatively by increasing hydrophobic interactions between them as well as with other residues (Fig. 3c). Taken together, the structural analyses of AKNc and AKDr suggest that hydrophobic CORE packing is not optimal in AKNc, and is important in thermal stability of fish AKs. Improvement in hydrophobic CORE packing increased thermal stability of AKNc. To investigate the role of hydrophobic packing around the three Val residues in temperature adaptation, we generated a series of AKNc mutants, in which the Val residues were substituted to Ile residues individually or collectively, and measured their T m values by CD spectroscopy (Table 1 and Fig. S1). The V28I mutation increased the thermal stability of AKNc considerably, as indicated by a 5.0 °C increase in T m compared with that in the wild-type (WT). The enhancement of thermal stability resulting from the other two individual Val-to-Ile mutations was relatively modest. The V118I and V173I mutations increased the T m of AKNc by 2.8 °C and 2.4 °C, respectively. However, the AKNc mutant with both the V118I and the V173I mutations exhibited an increase in T m of 7.6 °C relative to the WT AKNc. This value is greater than the sum of T m increases conferred by the V118I and V173I mutations individually, indicating a synergistic effect of the two mutations on the overall thermal stability. This is consistent with the structural analyses since the two residues are located close to each other (<4 Å) in the crystal structures (Fig. 2c).  Table 2. Data collection and refinement statistics a . a Values in parentheses are for the highest-resolution shell is the intensity of an individual measurement of the reflection and < I(h) > is the mean intensity of the reflection. c R cryst = ∑ h ||F obs | − |F calc ||/∑ h |F obs |, where F obs and F calc are the observed and calculated structure factor amplitudes, respectively. d R free was calculated as R cryst using 5% of the randomly selected unique reflections that were omitted from structure refinement.
ScIeNtIfIc REPORtS | 7: 16027 | DOI:10.1038/s41598-017-16266-9 The three Val-to-Ile mutations in combination resulted in the greatest thermal stability of AKNc (Table 1 and Fig. S1). The T m value of the V28I/V118I/V173I mutant was 11.3 °C higher than that of the WT AKNc, and was identical to that of AKDr, the most thermally stable homologue of the three tropical AKs. This observation suggests that the suboptimal hydrophobic packing around the three Val residues in the CORE domain is important for the reduced thermal stability of AKNc compared with that of its homologues from tropical fishes. We also tested the role of the hydrophobic CORE packing in thermal stability in the opposite direction. We produced an AKDr mutant in which Ile28, Ile118 and Ile173 residues were replaced with Val residues, and determined its T m value (Table 1 and Fig. S1). The reverse triple mutation significantly reduced the thermal stability of AKDr, as indicated by a decrease in T m of 8.9 °C relative to the WT enzyme, confirming the importance of the CORE packing in the overall stability of the fish AKs.
We also measured the thermal stabilities of S48A and T188K mutants of AKNc to test the effect of residue substitution at these positions. The three tropical AKs have Ala48 and Lys188, but AKNc has Ser48 and Thr188. The T m value of the S48A mutant was 2.2 °C higher than that of the WT AKNc, and the T188K mutation decreased the thermal stability of AKNc by 3.1 °C (Table 1). These results support the hypothesis that residues at these two positions may not be critical for overall thermal stability as they are not involved in intramolecular interactions connecting distant regions of the polypeptide, and suggest that the three Val residues play more important roles in the cold adaptation of AKNc.
To confirm that thermal stabilization caused by the Val-to-Ile mutations resulted from the optimized hydrophobic CORE packing, we determined the crystal structure of a V28I/V118I/V173I mutant of AKNc (Fig. S3). Data collection and refinement statistics are listed in Table 2. The overall tertiary structure of the mutant was essentially identical to that of the WT. The RMSD value of Cα atoms between the WT AKNc and the mutant was 0.15 Å. In the mutant structure, the conformations of the residues neighboring the mutated Ile28 residue were almost indistinguishable from those around Val28 in the WT AKNc structure (Fig. 4a). This allows the added terminal methyl group (Cδ1) of the Ile28 side chain to interact hydrophobically with other residues in the CORE domain-such as Val13, Cys25, Leu91, Leu116, Val186, and Ile190-indicating the enhancement of the hydrophobic packing by the V28I mutation. The crystal structure of the AKNc mutant also revealed that the V118I and V173I mutations optimized the hydrophobic packing in the CORE of AKNc (Fig. 4b). The conformation of Ile118 was essentially identical to that of Val118 in the WT structure, with the exception of the extra methyl group (Cδ1) in its side chain, which makes additional hydrophobic contacts with Val13, Gln24, Leu116, and Val186 in β1, α1, β4, and α9, respectively, in the mutant structure. In contrast, the side chain conformation of the mutated Ile173 residue was distinct from that of Val173 in WT AKNc. The χ1 dihedral angle along the bond between the Cα and Cβ atoms of Ile173 was rotated ~120° relative to that of Val173 in the WT AKNc structure. The flipped side chain of Ile173 interacts hydrophobically with residues in β4, β5, and α9, including Leu116, Arg171, Val182, Val186, and Ala189. The hydrophobic contact between the two mutated residues (Ile118 and Ile173) is enhanced by the extension and conformational change of their side chains. The structural comparison of the WT and mutant AKNc revealed that the three Val-to-Ile mutations optimized the packing of the hydrophobic interior of the CORE domain. Lys188 (e) in the crystal structure of AKDr. Ala48 and Lys188 are conserved among the three tropical AKs including AKDr, but not in AKNc. The side chains of Ala48 and Lys188 are exposed to the solvent, and do not interact closely with residues that are located distantly in the amino acid sequence.
Since our structural analyses are based on the Ap 5 A-bound structures, we also made T m measurements of the WT and the triple mutant AKs in the presence of Ap 5 A (Table 3 and Fig. S1). The addition of Ap 5 A caused significant T m increases for the AKs, indicating that the Ap 5 A binding stabilized the enzymes. Notably, the order in T m was maintained for the WT and mutant enzymes regardless of the presence of Ap 5 A, but the T m difference between them was reduced. For example, the V28I/V118I/V173I mutant of AKNc displayed higher T m than the WT AKNc by 11.3 °C without Ap 5 A, whereas the mutant was more thermally stable than the WT enzyme only by 4.5 °C in the presence of Ap 5 A. In the previous studies of bacterial AK variants, the effects of T m increase by applying multiple stabilization principles together were not strictly cumulative, and the magnitude of the T m enhancement varied depending on the backgrounds to which the stabilizing factors were added 24,26,43 . Thus, the results from the T m measurements with Ap 5 A seem to be consistent with the previous analyses, and, more importantly, confirm the validity of our structural analyses for the structural determinants of thermal stability. Optimization of hydrophobic CORE packing also affected catalytic function of AKNc. To examine the temperature dependence of the catalytic activity, we performed activity assays of WT AKNc and the mutant containing the three Val-to-Ile mutations (V28I/V118I/V173I) at various temperatures (Fig. 4c). The catalytic activity of the WT enzyme in terms of k cat peaked at 35 °C and decreased afterwards, compared to 45 °C for the mutant AKNc, which was more catalytically active than the WT AKNc at high temperatures (45 °C and 55 °C). The increase of the activity at high temperature could be a consequence of the enhanced thermal stability of the mutant AKNc. The inactivation of the enzymes at high temperatures most likely resulted from thermal denaturation. However, the mutant showed considerably decreased activity at low temperatures (5-35 °C) compared to the WT enzyme, which cannot be explained by the difference in thermal stability between the WT and mutant AKs. It seems that the improvement in hydrophobic CORE packing by the Val-to-Ile mutations increased the structural stability at high temperatures and decreased the catalytic activity at low temperatures. The stabilizing mutations might make the enzyme too rigid, and the dynamic motion required for its catalytic function might be impeded at low temperatures due to the reduced flexibility.
The activity assays of AKDr and its I28V/I118V/I173V mutant also showed consistent results (Fig. S4). The AKDr mutant exhibited increased catalytic activities at low temperatures (5 °C and 35 °C) compared to the WT enzyme, and the temperature of maximum activity decreased (35 °C). However, the magnitude of the activity change at low temperatures by mutation was not as significant as in AKNc. This suggests that AKDr contains extra structural feature(s) maintaining the rigidity of its structure in addition to the hydrophobic interactions involving the Ile residues. In the crystal structure of AKDr, we identified a salt bridge connecting between Arg171 and Glu192 (Fig. S5), which is substituted to Ala192 in AKNc. Consistently, the T m increase (11.3 °C) of AKNc by the three Val-to-Ile mutations was greater than the T m decrease (8.9 °C) of AKDr by the reverse triple mutation (Table 1), supporting the role of the salt bridge. Taken together, our results suggest that activity and stability of fish AKs are inversely correlated, and disruption of hydrophobic packing may be a structural mechanism of cold adaptation as it could increase catalytic activity at low temperatures.

Discussion
AKNc is useful for research on cold adaptation of psychrophilic enzymes for several reasons. The source organism of AKNc is the Antarctic fish N. coriiceps; therefore, the enzyme originated from a multicellular, eukaryotic psychrophile, whereas most previously characterized psychrophilic proteins were from psychrophilic microorganisms 4,5 . The conformational switching required for its enzymatic function also makes AKNc an attractive system to study the role of protein dynamics in cold adaptation as the maintenance of appropriate local and/or global motion is crucial for functioning at extreme temperatures. AK is a small protein that undergoes relatively large conformational changes upon substrate binding and product release (Fig. 1b), and has long been used as a model system for studying connections between structure, function, and dynamics [28][29][30]32,[44][45][46][47][48][49] .
The 'corresponding state' hypothesis first proposed by Somero postulates that homologous proteins originated from organisms living at different environmental temperatures have comparable flexibilities and activities at their physiologically relevant temperatures 20,50 . Although this hypothesis has widely been accepted by the scientific community, whether the reduced stability of psychrophilic proteins is a consequence of maintaining the conformational flexibility necessary for functional activity at low temperatures, or a result of a lack of evolutionary pressure, remains unclear 51 . In previous studies of bacterial AKs, the AK variants generated exhibited both thermal stability at high temperatures and sufficient catalytic activity at low temperatures, suggesting that activity at low temperatures can be achieved without sacrificing stability, and thus the low stability of psychrophilic proteins is not an adaptive trait 46,52 . However, in this study, the stabilizing Val-to-Ile mutations in AKNc reduced the activity at low temperatures, indicating that stability and activity are coupled, and the decreased thermal stability of AKNc might be required for sufficient catalytic activity in cold environments.
This contradiction likely results from structural differences in the LID domain of AKNc and its bacterial homologues. In AKNc, the LID is a short loop of less than 10 residues (Figs 1a and 2a), whereas the bacterial AKs have LID domains of >30 residues that include several β-strands (Fig. S6). The long LID domain is crucial for the function of bacterial AKs, as LID opening was the rate-limiting step in catalysis 47 , and conformational heterogeneity within the LID domain was important in functional adaptation 53,54 . Hence, it is conceivable that stability and activity are governed independently by different domains of bacterial AKs with the large LID domains, but not in short isoforms such as AKNc. In a previous study, swapping of the CORE domains of AKs from mesophilic and thermophilic bacteria affected T m values significantly, but did not affect the temperature dependence of activity, highlighting the spatial separation of stability and activity control in bacterial AKs 52 . Hence, for cold adaptation of bacterial AKs, residue substitutions in the LID domain are likely required to alter the LID dynamics, which are closely related to catalytic activity.   In the sequence alignment (Fig. 1a), the N-and C-terminal regions exhibited the greatest variability between AKNc and its homologues from tropical fishes. This observation suggests that these areas play more crucial roles in the temperature adaptation of AKs. The important role of the N-and C-terminal residues in overall thermal stability has already been noted for archaeal and bacterial AKs 23,24,44,55 . The chain folds of the AK homologues revealed that the first and last α helices (α1 and α9 in AKNc, respectively) in the amino acid sequence are located in close proximity 28,31,32 . Numerous polar and hydrophobic intramolecular interactions have been identified between the two terminal regions or involving residues from one of them 23,24,44 . In a previous study of archaeal AKs from the genus Methanococcus, chimeric AKs were constructed by exchanging the N-and C-terminal residues between mesophilic and hyperthermophilic homologues, and exhibited significant changes in T m values (~20 °C) compared to the WT proteins 55 . Experimental evolution for thermal stabilization of a mesophilic bacterial AK by Shamoo and co-workers resulted in the generation of stable AK variants containing mutations at six different positions, five of which were located within the first or last 30 residues of the amino acid sequence 56 .
The Ile-to-Val mutations in the CORE domain of AKDr resulted in a significant decrease in thermal stability (Table 1). Several previous studies of the stabilization of the mesophilic Bacillus subtilis AK (AKBs) have reported the importance of hydrophobic CORE packing in thermal stability 26 . Comparative analyses with a thermophilic homologue enabled identification of a residue substitution (T179M) that enhanced hydrophobic interactions in the CORE domain 23 , which increased thermal stability when introduced to AKBs 26 . The experimental evolution of AKBs also generated several mutations (Q16L, T179I, and A193V) 56 that stabilized the mesophilic target by improving the CORE packing 57 . Stable AKBs variants were previously designed based on a bioinformatic method of optimizing local structural entropy, an empirical descriptor of sequence heterogeneity 58 . The resulting AKBs mutants displayed increased apolar buried surface areas in the CORE domain, indicating the enhancement of hydrophobic contacts 43 . In a computational prediction followed by experimental validation, Wilson and co-workers tested 100 AKBs mutants and demonstrated that substantial thermal stabilization could be achieved by repacking of the hydrophobic CORE 59 .
In the present study, we discovered suboptimal hydrophobic packing in the CORE domain of the Antarctic fish AK. Comparative and mutational analyses demonstrated that imperfect hydrophobic CORE packing indeed results in reduced thermal stability and a shift in the temperature-activity profile. Our results suggest that modification of hydrophobic contacts is a key structural feature important for the cold adaptation of psychrophilic proteins, and may be used to engineer psychrophilicity in mesophilic enzymes.

Methods
Cloning, expression, and purification. Synthetic genes of the WT fish AK1 proteins were cloned into a pET28a vector with an N-terminal (His) 6 -maltose binding protein (MBP) tag and a tobacco etch virus (TEV) protease cleavage site. Mutant genes were generated by polymerase chain reaction (PCR) using mismatched primers. Escherichia coli BL21 (DE3) cells containing these constructs were cultured in LB medium at 37 °C until the optical density at 600 nm reached 0.7. Protein expression was then induced by the addition of 0.5 mM isopropyl-β-D-thiogalactopyranoside, followed by incubation at 17 °C for 16 h. The cells were harvested by centrifugation and resuspended in purification buffer (500 mM NaCl, 3 mM β-mercaptoethanol, 10% (w/v) glycerol, 20 mM Tris-HCl pH 7.0). After sonication and centrifugation, the supernatant was loaded onto a 5 mL HisTrap HP column (GE Healthcare, USA) equilibrated with purification buffer. The column was washed with purification buffer, and bound proteins were eluted by applying a linear gradient of imidazole (up to 500 mM). The (His) 6 -MBP tag was cleaved by TEV protease and separated using a HisTrap HP column. The proteins were further purified by size-exclusion chromatography using a HiLoad 16/60 Superdex 75 column (GE Healthcare, USA) equilibrated with size-exclusion chromatography buffer (300 mM NaCl, 3 mM dithiothreitol (DTT), 5% (w/v) glycerol, 50 mM HEPES pH 7.0).

Measurement of T m values.
T m values of AKs were determined by CD spectroscopy, as described previously 26 . CD traces at 220 nm were measured for 0.5 mg/mL AKs in 10 mM potassium phosphate pH 7.0 with or without 0.2 mM Ap 5 A. A Chirascan-plus CD Spectrometer (Applied Photophysics, UK) was used with a scanning rate of 1 °C/min. CD data were analyzed based on the protocol developed by John and Weeks 60 . Average values of three CD measurements at each temperature were differentiated to yield differential denaturation curves, which were fitted to parameters including T m using a two-state transition model.
Crystallization and structure determination. The WT and mutant AKNc proteins were crystallized under identical conditions. Their crystals were grown at 4 °C by the sitting-drop method from 18 mg/mL protein and 4 mM Ap 5 A in buffer (10 mM HEPES pH 7.0) mixed with an equal amount of reservoir solution (50% (v/v) polyethylene glycol 400, 200 mM lithium sulfate, 100 mM sodium acetate pH 4.5). The crystals were cryoprotected in the reservoir solution supplemented with 15% (v/v) ethylene glycol and flash-frozen in liquid nitrogen. The AKDr crystals were obtained at 20 °C by the sitting-drop method from 20 mg/mL protein and 4 mM Ap 5 A in buffer (10 mM HEPES pH 7.0) mixed with an equal amount of reservoir solution (2.5 M ammonium sulfate, 0.1 M sodium acetate pH 4.6). The crystals were cryoprotected in the reservoir solution supplemented with 20% (v/v) ethylene glycol and flash-frozen in liquid nitrogen.
Diffraction data from the AKNc and AKDr crystals were collected at 100 K at the beamlines 5 C and 7 A of the Pohang Accelerator Laboratory. Diffraction images were processed with HKL2000 61 . PHASER was used for molecular replacement phasing 62 . The structure of human AK1 (Protein Data Bank code 1Z83) was used as a starting model for the WT AKNc. Molecular replacement solutions for AKDr and the AKNc mutant were found with the WT AKNc structure. The final structures were completed using alternate cycles of manual fitting in COOT 63 and refinement in REFMAC5 64 . The stereochemical quality of the final models was assessed using MolProbity 65 .
ScIeNtIfIc REPORtS | 7: 16027 | DOI:10.1038/s41598-017-16266-9 Temperature-dependent activity assay. AK activity was measured at multiple temperatures in the direction of ATP formation as described previously, with minor modifications 52 . The enzymatic reaction was started by the addition of AK (1.1 ng/mL final concentration) to the reaction mixture (2.5 mM ADP, 1 mM glucose, 0.4 mM NADP + , 100 mM KCl, 2 mM MgCl 2 , 50 mM HEPES pH 7.4). After incubation at the indicated temperatures for 5 min, the reaction was stopped by the addition of 0.5 mM Ap 5 A. The amount of ATP produced by the reaction was determined by ATP-dependent NADP + reduction to NADPH using hexokinase and glucose-6-phosphate dehydrogenase at room temperature. Average values of three independent measurements were reported with standard errors. Data availability. The atomic coordinates and structure factors were deposited in the Protein Data Bank 66 .