Activity-stability trade-off observed in variants at position 315 of the GH10 xylanase XynR

XynR is a thermostable alkaline GH10 xylanase, for which we have previously examined the effects of saturation mutagenesis at position 315 on enzyme alkaliphily, and found that at pH 10, the activities of variants could be ordered as follows: T315Q > T315S = T315N > T315H = wild-type XynR (WT) > 15 other variants. In this study, we sought to elucidate the mechanisms underlying the variable activity of these different variants. Crystallographic analysis revealed that the Ca2+ ion near position 315 in WT was absent in the T315Q variant. We accordingly hypothesized that the enhancement of alkaliphily in T315Q, and probably also in the T315H, T315N, and T315S variants, could be ascribed to an activity-stability trade-off associated with a reduction in stability due to the lack of this Ca2+ ion. Consistent with expectations, the alkaline resistance of T315H, T315N, T315Q, and T315S, evaluated through the pH-dependence of stability at 0 mM CaCl2 under alkaline conditions, was found to be lower than that of WT: the residual activity at pH 11 of WT was 78% while those of T315H, T315N, T315Q, and T315S were 0, 9, 0, and 43%, respectively. In addition, the thermostabilities of these four variants, as assessed using the denaturing temperatures (Tm) at 0 mM CaCl2 based on ellipticity at 222 nm in circular dichroism measurements, were lower than that of WT by 2–8 °C. Furthermore, the Tm values of WT and variants at 5 mM CaCl2 were higher than those at 0 mM CaCl2 by 6–11 °C. Collectively, our findings in this study indicate that mutation of the T residue at position 315 of XynR to H, N, Q, and S causes an increase in the alkaliphily of this enzyme, thereby reducing its stability.


Refinement of the structure of T315Q
We made crystallographic analysis of T315Q.Table 1 summarizes data collection and refinement statistics.The space group of the crystal was C2.The structure was refined at 1.90 Å resolution.The whole structure of T315Q exhibited no difference compared to WT 13 with rmsd of 0.65 Å for 353 Cα atoms.Figure 1A shows the mutationsite structure of T315Q.Phenix polder omit map shows that T315 was replaced with Q in T315Q.WT had three Ca 2+ ions 13 , but T315Q had one Ca 2+ ion. Figure 1B shows the comparison of mutation-site structures of WT and T315Q.In WT, the Ca 2+ ion was observed, which was surrounded by four ligating residues (D312, D318, D331, and Y273), suggesting that this Ca 2+ ion plays certain roles in the stability of active site.T315Q lacked this Ca 2+ ion.In WT, T315 did not interact with the Ca 2+ ion.In T315Q, the side chain of Q315 protrudes inside, resulting in that the distance of Nε2 from the position where the Ca 2+ ion located in WT was 1.8 Å.This suggested that such unfavorable distance (1.8 Å) leaded to the steric hindrance between the Nε2 of Q315 and the Ca 2+ ion, resulting in the lack of the Ca 2+ ion in T315Q.The Nε2 of Q315 forms hydrogen bonds with Oδ1 of D318, O of D331 and O of D312 instead of the coordination bond of Ca 2+ ion in WT, suggesting partial stabilization of this site.
Generally, mutations which increase activity are accompanied with decrease in stability and vice versa.This relationship between activity and stability was well known as activity-stability trade-offs 14,15 .To address this hypothesis, we compared the stabilities of WT and variants in the subsequent studies.

pH dependence of stability
We examined pH dependences of stability of WT and four variants (T315H, T315N, T315Q, and T315S).The enzymes were incubated at various pHs and 37 °C for 24 h.After incubation, the activities to hydrolyze beechwood xylan were measured at pH 8.0 and 37 °C (Fig. 2).T315Q and T315H exhibited markedly narrower bell-shaped pH-dependence of stabilities than WT.T315N and T315S also exhibited narrower bell-shaped pHdependences of stability than WT.The residual activity at pH 11 of WT was 78% while those of T315H, T315N, T315Q, and T315S were 0, 9, 0, and 43%, respectively.These results suggested that the amino acid residue whose side chain has an amido or imidazole group at position 315 makes XynR less alkaline-resistant.

Thermal denaturation
Scheme 1 has been used for evaluating enzyme stability.
where N and D represent the native and denatured species, respectively.In Scheme 1, the stability of protein is assessed by ΔG° that represents the difference in G° between the native and denatured states at a certain temperature or T m , the temperature at which F u is 0.5 in Eq. ( 1).
The CD spectra of WT and four variants (T315H, T315N, T315Q, and T315S) at pH 8.0 and 25 °C exhibited negative ellipticities at around 200-250 nm with minimum values around 222 nm 12 , indicating the mutation at position 315 did not elicit drastic structural changes.Figure 3A-E showed thermal denaturation of WT and variants by monitoring [θ] 222 in the range of 25-90 °C.The denaturation curves of WT and variants showed apparent two-state model.However, this thermal denaturation was not reversible, suggesting that Scheme 1 was not fully applicable.The T m values at 0 and 5 mM CaCl 2 of WT and variants are shown in Table 2.They were in the order of WT > T315N ≈ T315S > T315H ≈ T315Q, indicating that the mutation at position 315 decreased the stability of XynR.The T m values at 5 mM CaCl 2 were 6.4-10.6 °C higher than those at 0 mM CaCl 2 .This suggested that thermal treatment promotes the dissociation of the Ca 2+ ion from the active site, while addition of excess Ca 2+ ions promotes the binding.
It is reported that the molten globule state is an intermediate state that has lost the majority of tertiary structure but retains a significant amount of secondary structures 16 , suggesting that the T m values might be lower than those in Table 2 if the values were assessed by the other methods such as fluorescence and differential scanning calorimetry.where N, D, and PD represent the native, denatured, and partially denatured species, respectively.In Scheme 2, stability of protein is assessed by ΔG° ‡ that represents the difference in G° ‡ between the native and transition states and is obtained from the k obs using Eq. ( 3).WT and variants were incubated at pH 8.0 and 58-72 °C for 2-16 min, and the remaining activities were determined at pH 8.0 and 37 °C (Fig. 4A-E, left panel).The natural logarithm of the remaining activities of WT and variants plotted against the incubation time gave linear relationships at all temperatures examined (Fig. 4A-E, right panel), indicating that the inactivation followed pseudo-first-order kinetics.The k obs values and half-life (τ 1/2 ) values of WT and variants at each temperature are summarized in Table S1.They increased with increasing temperatures.The temperatures at which the k obs values were 0.05-0.10min -1 in the order of WT > T315N ≈ T315S > T315H ≈ T315Q, indicating again that the mutation at position 315 decreased the stability of XynR.
Figure 4F shows the Arrhenius plot of k obs .Linear relationship was obtained between the logarithmic value of k obs and 1/T.The thermodynamic parameters for thermal inactivation of WT and variants at 65 °C, which are obtained using Eqs.( 3)-( 5), are summarized in Table 3.The ΔG° ‡ values that reflect the protein stability were in the order of WT > T315N ≈ T315S > T315H ≈ T315Q.All variants exhibited decreased ΔH° ‡ and ΔS° ‡ values, indicating that the decreased thermal stability was due to the decrease in ∆H° ‡.Notably, the magnitude

Effects of Ca 2+ binding on stability
To explore the role of the Ca 2+ ion on the mutational effects at position 315, the thermal inactivation of WT and variants at 62 °C in the presence of various CaCl 2 concentrations (0-5 mM) was investigated (Fig. 5, Fig. S2).
The k obs values are shown in Fig. 5F and summarized in Table S2.The k obs values of WT and T315S were constant at all CaCl 2 concentrations examined.The k obs values of T315N were constant at all CaCl 2 concentrations except for 0 mM.The k obs values of T315H and T315Q decreased and their τ 1/2 values increased with increasing CaCl 2 concentrations (0-0.1 mM) and were constant at 0.1-5 mM CaCl 2 .The CaCl 2 concentration-independent k obs values (min −1 ) were 0.001-0.004for WT, 0.020-0.054for T315H, 0.007-0.016for T315N, 0.029-0.062for T315Q, and 0.0024-0.0086for T315S (Table S2), indicating that the value was in the order of WT < T315S < T 315N < T315H < T315Q.
To explore the role of this extended loop on the substrate binding and catalytic activity, we made crystallographic analysis of WT complexed with xylobiose.As shown in Table 1, the space group of the crystal was  7B shows the comparison of WT and T315Q.In the region (residue 311-331) with the largest deviation between T315Q and WT (Fig. 6), three residues (D312, D318, and D331) are involved in the Ca 2+ binding, and two residues are involved in the binding to xylobiose.R320 interacts with O5 of subsite + 1 xylose and O3 of subsite + 2 17 , and is located near the acidic and basic catalytic residues (E150 and E256, respectively) with 8.0 and 8.8 Å, respectively.These evidences suggest that the mutation of T315 to Q deviates the position of R320, affecting the dissociation of E150, leading to increase in alkaliphily of T315Q.
In conclusion, the alkaliphilic XynR variant T315Q lacked the Ca 2+ ion near position 315 by keeping the interaction with three of four ligating residues with the Ca 2+ ion.Our results suggested that T315Q, and presumably T315H, T315N, and T315S, obtained higher alkaliphily by decreasing stability accompanied by the loss of this Ca 2+ ion and the resulting loop (residues 311-331) deviation.

Expression and purification of WT and variants
One hundred milliliter of LB broth containing 50 μg/mL ampicillin was inoculated with the glycerol stock of the transformed E. coli BL21 (DE3) and incubated at 37 °C with shaking.When OD 660 reached 0.6-0.8,isopropylβ-D-thiogalactopyranoside (IPTG) (25 μL of 500 mM) was added, and growth was continued at 30 °C for 24 h.The cells were harvested by centrifugation and suspended with 50 mL of 20 mM phosphate-NaOH buffer (pH 8.0) (buffer A) and disrupted by sonication.After centrifugation, the supernatant was collected.Solid (NH 4 ) 2 SO 4 was added to the supernatant to be 50% saturation.The pellet resulted was collected by centrifugation, dissolved in buffer A containing 0.5 M NaCl (buffer B), and dialyzed against buffer A. The crude enzyme solution thus obtained was applied to a HisTrap™ HP column (GE Healthcare, Buckinghamshire, UK) equilibrated with buffer B. After the was wash with 50 mL of buffer B, XynR was recovered by the elution with buffer B containing 0.5 M imidazole.Each fraction (5 mL) was assessed to contain WT or variants by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE).Active fractions were concentrated after desalting.Purified enzyme solution was stored at 4 °C.

Crystallization and structural determination
Crystals were obtained in the following conditions: (i) ligand-free T315Q: 1 μL of 12.7 mg/mL enzyme solution in 20 mM phosphate-NaOH buffer (pH 8.0) was mixed with 1 μL of reservoir solution (0.2 M ammonium iodide, 26% w/v polyethylene glycol (PEG) 4000, pH 5.9) and was equilibrated against 100 μL of reservoir solution at 20 °C, using the sitting drop vapor-diffusion method in a 96-well plate (Intelli-Plate, Art Robbins Instrument, Sunnyvale, CA).Rectangle crystals were obtained after a few weeks.(ii) WT complexed with xylobiose: 1 μL of 13.0 mg/mL enzyme solution in 0.35 M xylobiose and 20 mM tris-hydroxymethyl aminomethane (Tris)-HCl buffer (pH 8.0) was mixed with 1 μL of reservoir solution (0.02 M CaCl 2 , 0.1 M sodium acetate, 22% v/v Crystals were flash-cooled in nitrogen gas stream at 100 K. Diffraction data were collected at the BL26B1 station of SPring-8, Sayo, Hyogo, Japan with the approval of JASRI (proposal nos.2021B2760 and 2023B2733), after the diffraction was checked by an in-house detector system of Bruker D8 Venture.The collected diffraction data were processed with XDS 18 .Molecular replacement was conducted using the structure of WT 13 as a search model (PDB, 7CPK).Structure refinement was conducted using COOT 19 and PHENIX 20 .

Hydrolysis of beechwood xylan
The activity was measured as described previously 9,10 .Briefly, the reaction was initiated by mixing 10 μL of enzyme solution and 90 μL of substrate solution (10 mg/mL beechwood xylan in 100 mM phosphate-NaOH buffer at pH 8.0) both pre-incubated at 37 °C.The reaction solution was incubated at 37 °C, and 100 µL of DNS solution (0.5% w/v DNS, 1.6% w/v NaOH, 30% w/v potassium sodium tartrate) was added to stop the reaction at predetermined times.After incubating at 100 °C for 15 min and at 0 °C for 3 min, 80 µL of the solution and 120 µL of water were mixed, and A 540 was measured with a multimodal plate reader EnSight (PerkinElmer, Waltham, MA).The standard curve was made using xylose.The concentrations of reducing sugars were estimated from the standard curve.The initial reaction rate was estimated from the time-course for production of reducing sugars.pH treatment of WT and variants was initiated by mixing 5 μL of enzyme solution (1.5 μM) and 45 μL of buffer solution (100 mM acetate-sodium acetate buffer (pH 3.5-5.5),100 mM phosphate-NaOH buffer (pH 6.0-8.5), or 100 mM carbonate-bicarbonate buffer (pH 9.0-11.0)).The treatment continued at 37 °C for 24 h.Then, activity of the pH-treated enzyme solution was measured as described above.

Circular dichroism (CD) measurement
The CD spectra of WT and variants were measured using a J-820 spectropolarimeter (Jasco, Tokyo, Japan) with a Peltier system of cell temperature control under the following conditions: spectral range 200-250 nm; 100 mdeg sensitivity; 0.2 nm resolutions; 1 s response time; 10 nm min −1 scan rate; and 5 accumulations.CD spectra were recorded at 37 °C using a 2-mm cell.The concentration of each enzyme was 1.0 µM.CD spectra were processed with a Jasco software, and finally expressed in mean-residue molar ellipticity units, [θ] (deg cm 2 dmol −1 ).
where A O is the observed [θ] 222 of WT or variants at various temperatures, and A N and A D are θ 222 of native and denatured enzymes, respectively.The temperature at which F u is 0.5 is defined as an apparent denaturing temperature (T m ).
Then, the beechwood xylan-hydrolyzing activity was determined as described above.The first-order rate constant of thermal inactivation (k obs ) was evaluated by plotting natural logarithm of the residual activity against the duration time of thermal treatment.The activation energy for the thermal inactivation (E a ) and the standard Gibbs energy change of activation for thermal inactivation (ΔG° ‡) were determined according to Arrhenius plot (Eqs.2, 3), respectively.

(Figure 1 .
Figure 1.Ca 2+ -binding site in the active site.(A) T315Q.Phenix polder omit of Q315 is contoured at 4σ. (B) Superposition of T315Q and WT.The peptides of T315Q and WT are colored in yellow and cyan, respectively.The Ca 2+ is shown as a grey sphere.The number indicates the distance (Å).

Figure 2 .Figure 3 .
Figure 2. Effect of pH on stabilities of WT and variants.The enzymes (0.15 μM) were incubated at various pHs and 37 °C for 24 h.The incubation buffers were 100 mM acetate-sodium acetate buffer (pH 3.5-5.5),100 mM phosphate-NaOH buffer (pH 6.0-8.5), and 100 mM carbonate-bicarbonate buffer (pH 9.0-11.0).Then, hydrolysis reaction of beechwood xylan was carried out at 37 °C with the enzyme and initial substrate concentrations of 0.015 μM and 9 mg/mL, respectively.Residual activity indicates the value compared to that before the incubation at pH 3.5-11.0.Error bars indicate SD values of triplicate determinations.

Figure 4 .
Figure 4. Thermal inactivation of WT and variants.(A-E) The enzymes (1.5 μM) were incubated in 100 mM HEPES-NaOH buffer (pH 8.0) at 58-72 °C for specified time.Beechwood xylan-hydrolysis activities were measured at 37 °C after thermal incubation.Residual activity was expressed as the relative value to that before the incubation at 58-72 °C.Residual activities were plotted against incubation time.(F) Arrhenius plot of k obs values.The natural logarithms of k obs values were plotted against the reciprocal of absolute temperature of thermal inactivation.

Figure 5 .
Figure 5.Effect of CaCl 2 concentrations on thermal inactivation of WT and variants.The enzymes (1.5 μM) were incubated in 100 mM HEPES-NaOH buffer (pH 8.0) and 0-0.8 mM CaCl 2 at 62 °C for specified time.Beechwood xylan-hydrolysis activities were measured at 37 °C after thermal incubation.(A-E) Residual activities of WT and four variants were plotted against incubation time.(F) First-order rate constants of thermal inactivation (k obs ) of WT and four variants were plotted against CaCl 2 concentrations of 0-1 mM.

Figure 7 .
Figure 7. Active-site structure of XynR complexed with xylobiose.(A) WT.The peptide is colored in green, the xylobiose is colored in yellow, and the Ca 2+ is shown as grey sphere.Phenix polder omit of xylobiose is contoured at 4σ. (B) Superposition of WT and T315Q (left and right stereo drawing).The peptides of WT and T315Q are colored in yellow and cyan, respectively.The Ca 2+ is shown as yellow sphere.The number indicates the distance (Å).

Table 1 .
Data collection and refinement statistics of XynR.

Table 2 .
T m of WT and variants.Averages of duplicate determinations are shown.Values in parentheses indicate those relative to WT.