DOI: 10.5061/dryad.ncjsxkt6b
title: Data from: Characterisation of a cold-adapted, thermostable glucokinase from psychrophilic <em>Pseudoalteromonas</em> sp. AS-131 reveals how the enzyme achieves high thermal stability without loss of cold adaptation

description: Microorganisms living in cold environments such as the Antarctic and deep
 sea usually possess cold-adapted enzymes, which are known to have high
 catalytic efficiency and low stability owing to their flexible structures.
 Research on cold-adapted enzymes has not progressed much due to the
 challenge of these enzymes being less stable. However, several
 cold-adapted enzymes with high thermal stability have recently been
 reported. In this study, we investigated the biochemical properties of
 glucokinases from the psychrophilic Pseudoalteromonas sp. AS-131 (PsGK)
 isolated from the Antarctic Ocean and mesophilic Escherichia coli (EcGK).
 We demonstrated that PsGK is a cold-adapted enzyme with high thermal
 stability. A comparison of the crystal structures and spectroscopic
 studies revealed that PsGK has an additional disulfide bond connecting the
 N- and C-termini. To test whether this bond is important for stability, we
 prepared a PsGK variant by removing the bond and observed a significant
 reduction in thermal stability. In addition, the introduction of the
 artificial disulfide bonds in homologous positions in EcGK increased the
 thermal stability without the reduction of maximum activity. These results
 confirmed that the introduction of a disulfide bond at the proper
 position, such as the connection of the N- and C-termini, significantly
 improved stability without changing the nature of enzymes. Our findings
 propose a new strategy that will contribute to the industrial application
 of enzymes.
description: <strong>Cloning, expression, and
 purificatio</strong>n It was assumed that the
 genes of <em>Pseudoalteromonas</em> sp. AS-131 would be highly
 conserved with those of <em>Pseudoalteromonas
 haloplanktis</em> TAC125 since one of the DNA sequences, alkaline
 protease gene (<em>fpa</em>), from AS-131 exhibited 99%
 identity to the <em>fpa</em> gene of TAC125. Subsequently, the
 nucleotide sequence of the glucokinase gene in AS-131 was identified using
 the <em>phglk</em> sequence from TAC125. The protein encoded
 by this gene (UniProt ID: H7CHS4) was referred to as PsGK in this study.
 For the comparison with PsGK, the genome sequence of <em>Escherichia
 coli</em> K-12 was obtained from GenBank, and the glucokinase gene
 (<em>glk</em>) was identified. The protein encoded by
 <em>glk</em> (UniProt ID: P0A6V8) was referred to as
 EcGK. PsGK and EcGK DNAs were cloned into the
 expression vector pET-16b by homologous recombination using a yeast-based
 <em>in vivo</em> cloning method. These DNAs were amplified by
 PCR using the following primers: a forward primer,
 5′-TGTTTAACTTTAAGAAGGAGATATACC<strong>ATG</strong>AGCTTACACTCTTCTGCC-3′, and a reverse primer, 3′- CGCAGCTTCCTTTCGGGCTTTGTTAGCAGC<strong>TTA</strong>TTCCTGCTTACTATTATGCAAACAC-5′ (NOTE: underlined and bold sequences represent homologs of the pET-16b vector start and stop codon, respectively). The amplified DNA fragment and pET-16b vector digested with <em>Bam</em>HI were mixed, and <em>Saccharomyces cerevisiae</em> YPH499 competent cells were transformed. Recombinant plasmid DNA was isolated from <em>S. cerevisiae</em> using the QIAprep Miniprep Kit (Qiagen). The rescued plasmid was transformed into <em>Escherichia coli</em> (<em>E. coli</em>) DH5α for propagation. The recombinant plasmid was transferred to <em>E. coli</em> DH5α. The recombinant plasmid DNA isolated from <em>E. coli</em> DH5α was transformed into <em>E. coli</em> BL21 (DE3) for expression. Similarly, the gene encoding EcGK was amplified by PCR from the genomic DNA of <em>E. coli</em> K-12 obtained from GenBank and cloned into the expression vector pET-16b. In addition, PsGK C156S, EcGK C20S, EcGK C60S, and EcGK C65S, for the identification of cysteines exposed to solution, and PsGK C325S, EcGK H312C, EcGK C20S C65S H312C, EcGK C20S C65S L313C, and EcGK C20S C65S S309C, for CD spectroscopy, were synthesised by GenScript Biotech and cloned into the expression vector pET-16b digested with <em>Nco</em>I and <em>Bam</em>HI. <em>E. coli</em> BL21 (DE3) was purchased from Funakoshi and transformed with the expression vector, inoculated into LB medium containing 50 µg/mL of ampicillin, and grown at 37°C until OD<sub>660nm</sub> was 0.6-1.0. Isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added to the culture medium (a final concentration of 1.0 mM), and protein expression was induced at 20°C for 24 h. All conditions for culturing were optimised to achieve high expression of GK. The harvested cells were suspended in 20 mM Tris-HCl buffer (pH 7.6) containing 10 mM MgCl<sub>2</sub>, followed by the addition of dithiothreitol (DTT), phenylmethylsulfonyl fluoride, and lysozyme were added to the cell suspension(final concentrations of 1.0 mM, 1.0 mM, and 0.08%, respectively), and incubated on ice for approximately 20 min. Sodium deoxycholate was added at the final concentration of 0.2% and stored at -80°C. The cell suspension was then disrupted by freezing and thawing. Streptomycin was added to the suspension at a final concentration of 2.0% to remove nucleic acids. The suspension was gently stirred and centrifuged at 10,000 rpm for 20 minutes. The supernatant was collected and subjected to ammonium sulfate fractionation. The active fractions of 20-50% and 30-60% ammonium sulfate saturation for PsGK and EcGK, respectively, were collected and dialysed with 20 mM Tris-HCl buffer (pH 7.6) containing 10 mM MgCl<sub>2</sub> and 5 mM β-mercaptoethanol. The dialysed samples were loaded onto the anion-exchange chromatography using HiTrap Q HP (Cytiv, a) and the bound protein was eluted with a linear gradient of KCl (0 to 300 mM in 20 mM Tris-HCl buffer (pH 7.6) containing 10 mM MgCl<sub>2</sub> and 5 mM β-mercaptoethanol). The expression and purity of PsGK and EcGK were assessed by SDS-PAGE. The molecular weights of the purified PsGK WT and EcGK WT were estimated by gel filtration using a HiLoad 16/60 Superdex 200 prep grade column (Cytiva). Protein concentration was determined by measuring the absorbance at 280 nm (ε<sub>280nm</sub> = 21.4 mM/cm and 28.4 mM/cm for PsGK and EcGK, respectively). <strong>Activity assay</strong> The glucokinase activity was measured using a two-step reaction. For the first step, a reaction mixture containing 10 µL of enzyme and 290 µL of substrate (5 mM glucose and 2 mM ATP) in 20 mM Tris-HCl buffer (pH 7.6) containing 10 mM MgCl<sub>2</sub> and 1 mM DTT was incubated for 5 min at various temperatures (1-80°C). The reaction was stopped by the addition of 15 µL of 50% (w/w) trichloroacetic acid and neutralised by the addition of 30 µL of 2 M Tris. In the second step, a reaction mixture from the first step and 50 µM d-glucose 6-phosphate dehydrogenase (G6PD) in 20 mM Tris-HCl buffer (pH 7.6) containing 10 mM MgCl<sub>2</sub> and 1.2 mM NADP<sup>+</sup> was incubated for 20-30 min at room temperature. The amount of glucose 6-phosphate produced by glucokinase was estimated by measuring the absorbance of NADPH, the product of the second step, at 340 nm using a microplate reader (Thermo Scientific). One unit of glucokinase activity was defined as the enzymatic activity required to phosphorylate 1.0 µmol of glucose to 1.0 µmol glucose 6-phosphate per minute under the described conditions. <strong>Thermal stability</strong> The thermal stability of the glucokinases was determined by measuring residual activity and CD spectroscopy. Residual activities were determined by the following two experiments. In the first experiment, the enzyme solutions were heated at various temperatures ranging from 45 to 70°C for up to 60 min. At each temperature, samples were taken every 10 min, rapidly cooled on ice. Residual activity was determined under the optimum reaction conditions for each enzyme, with the initial activity at zero time defined as 100%. The <em>k</em><sub>d</sub> was determined from the slope of a plot of the log of residual activity against time. In the second experiment, the residual activities were measured at optimum conditions after pre-incubation at various temperatures for 10 min in a 20 mM Tris-HCl (pH 7.6) buffer containing 10 mM MgCl<sub>2</sub>, evaluated with the specific activity at optimum conditions as 100%. To estimate the <em>T</em><sub>50</sub>, the residual activity measured over time was fitted to a logistic regression model. CD measurements were performed using a Jasco J-1500 spectropolarimeter at the Institute for Molecular Science, Okazaki, equipped with a Peltier cell holder and a PTC-510 temperature controller using a quartz cell with 0.1 mm path length (Jasco). To assess the secondary structure, CD spectra were collected at 20°C in the wavelength range of 200-260 nm. The measurement parameters were set as follows: 0.1 nm data pitch, 20 nm/min scanning speed, 1 nm band width, 1 s response time, and 100 mdeg sensitivity. The enzymes were prepared in 10 mM potassium phosphate buffer (pH 7.6) at a concentration of 5 µM. For thermal denaturation analysis, the ellipticity at 222 nm was monitored while gradually increasing the temperature from 5 to 90°C, with data recorded at 0.2°C intervals under continuous temperature control by the Peltier device. The unfolding curves were analysed using a sigmoidal curve function, according to Rünzler et al. Where <em>θ<sub>T</sub></em> is the ellipticity at temperature <em>T</em>, <em>m<sup>T</sup></em> is the slope of the curve within the transition region, and the inflection point of the curve represents the melting temperature <em>T<sub>m</sub></em>. The <em>T</em><sub>m</sub> values were calculated from these thermal melting curves. The fitting errors of <em>T</em><sub>m</sub> were used as the standard errors of the measurements. At each temperature, <em>b<sub>N</sub></em> and <em>b<sub>D</sub></em> can be extrapolated from the pre- and post-transition baselines, (<em>m<sub>N</sub></em> × <em>T</em> - <em>b<sub>N</sub></em>) and (<em>m<sub>D</sub></em> × <em>T</em> - <em>b<sub>D</sub></em>), respectively. The fraction of unfolded proteins was calculated by subtracting the baseline values. <strong>Kinetic parameter</strong> <em>K</em><sub>m</sub> and <em>k<sub>c</sub></em><sub>at</sub> of PsGK and EcGK were measured at 1, 25, and 40°C in 20 mM Tris-HCl (pH 7.6) buffer containing 10 mM MgCl<sub>2</sub>, 5 mM glucose, 2 mM ATP, 1 mM DTT, 1.2 mM nicotinamide adenine dinucleotide phosphate (NADP<sup>+,</sup>) and 50 µM G6PD. The concentrations of glucose and ATP varied from 0.17 to 1.0 mM. <em>K</em><sub>m</sub> and <em>k<sub>c</sub></em><sub>at</sub> for each substrate were determined by steady-state experiments performed at 1, 25, and 40°C in assay buffer containing 20 mM Tris-HCl (pH 7.6), 10 mM MgCl<sub>2</sub>, 1 mM DTT, 1.2 mM NADP<sup>+</sup>, 50 µM G6PD, and various concentrations of glucose and ATP, using Lineweaver-Burk plots based on Michaelis-Menten enzyme kinetics. The values represent mean values ± standard deviation of three independent experiments in Table 1. <strong>Thermodynamic parameter</strong> The <em>E</em><sub>a</sub> of the reaction catalysed by PsGK and EcGK were calculated by measuring the slope of the Arrhenius plot, which was made based on the <em>k</em><sub>cat</sub> values at 1-40°C. <em>k</em><sub>cat</sub> values used in the Arrhenius plot were calculated based on the reaction rates measured at given temperatures. The thermodynamic activation parameters of PsGK and EcGK at 25°C were calculated using respective equations. <strong>DTNB method</strong> Ellman’s reagent, DTNB, has the ability to bind to the free thiol groups by cleaving its own disulfide bond and releasing 5-mercapto-2-nitrobenzoic acid (TNB). Spontaneously, TNB is ionised to the TNB<sup>2-</sup> dianion in water at neutral and alkaline pH. As DTNB is reacted with the free thiol group of cysteine at a one-to-one ratio, the concentration of free thiol groups can be quantified by measuring the absorbance of TNB<sup>2-</sup>. Furthermore, DTNB reacts slowly or does not react with free cysteine residues inside the enzyme, depending on the degree of structural flexibility. To avoid the reaction of DTNB with the reductant, β-mercaptoethanol in the samples was removed using a desalting spin column (APRO Science group). DTNB was dissolved in 10 mM phosphate buffer (pH 6.5) to a final concentration of 4 mM. The mixing ratio of enzyme, 0.1 M Tris-HCl buffer (pH 8.0) containing 1 mM ethylenediaminetetraacetic acid, was adjusted by the number of expected free cysteine residues so that the absorbance at 412 nm of TNB<sup>2-</sup> (λ<sub>max</sub> = 412 nm, ε = 14,150 M<sup>-1</sup>cm<sup>-1</sup>) was approximately 0.2 per one cysteine residue. DTNB was added at a molar amount 20-fold greater than that of the enzyme. The reaction mixture was initiated to react for 1 min, and then started to record the absorbance was recorded at 412 nm for 5-10 min. After 3-12 h, the absorbance was measured at 412 nm. <strong>Molecular dynamics</strong> MD simulations were performed using the AMBER16 program package. The protein structure was obtained from the Protein Data Bank (PDB ID: 1Q18), and specific amino acid residues were replaced with cysteines (S309C and L313C) to allow disulfide bond formation. The disulfide bond was explicitly defined using the bond command in LEaP. The ff14SB all-atom force field was applied. Water was modeled by the TIP3P potential, and a protein molecule was surrounded by a periodic octahedral box of TIP3P water molecules. Na+⁺ counterions were added to neutralise the system. All Lennard-Jones interactions were cut off at a distance of 10 Å, and long-range electrostatic interactions were calculated using a particle mesh Ewald method. The MD simulations were set up using the following protocol. First, 1000 steps of energy minimisation were performed to remove close van der Waals contacts present in the initial structure and to allow the formation of hydrogen bonds between solvent molecules and the protein. In this stage, the protein was kept fixed, and only the positions of water molecules were minimised. In the second step, an unrestrained energy minimisation for 2,500 steps was performed, in which the entire system was minimised. As the third step, the system was then gradually heated to 300 K over 20 ps under constant volume conditions. The temperature was controlled using Langevin dynamics with a collision frequency of 1.0 ps⁻¹. To constrain bonds involving hydrogen atoms, the SHAKE algorithm was employed, and the time integration step was set to 2 fs. After heating, the system was simulated for 50 ns under constant temperature (300 K) and pressure (1 atm) conditions, using a time step of 2 fs. The distance between the SG of the cysteine residues forming the disulfide bond was extracted from all simulation frames and analysed to evaluate its time-dependent variation.
description: # Data from: Characterisation of a cold-adapted, thermostable glucokinase
 from psychrophilic *Pseudoalteromonas* sp. AS-131 reveals how the enzyme
 achieves high thermal stability without loss of cold adaptation Dataset
 DOI: [10.5061/dryad.ncjsxkt6b](https://doi.org/10.5061/dryad.ncjsxkt6b) ##
 Description of the data and file structure ### Files and variables ####
 File: Fig2a_specific_activity_EcGK_WT.csv
 **Description:** **Characterisation of glucokinase from** **glucokinase
 from** ***Escherichia coli*** **(EcGK) wild type (WT)** ##### Variables *
 Temp:0-65 * U/mg:0-40 * SD:The values represent mean values ± standard
 deviation of three independent experiments (n = 3). #### File:
 Fig2b_residual_activity_EcGK_WT_50℃.csv
 **Description:** **Characterisation of glucokinase
 from*Pseudoalteromonas*** **sp. AS-131 (PsGK) wild type (WT)** #####
 Variables * Time (min):0-60 * Log of residual activity:0-2 * SD:The values
 represent mean values ± standard deviation of three independent
 experiments (n = 3). #### File: Fig2b_residual_activity_EcGK_WT_55℃.csv
 **Description:** **Characterisation of glucokinase from** **glucokinase
 from** ***Escherichia coli*** **(EcGK) wild type (WT)** ##### Variables *
 Time (min):0-60 * Log of residual activity:0-2 * SD:The values represent
 mean values ± standard deviation of three independent experiments (n = 3).
 #### File: Fig2b_residual_activity_EcGK_WT_45℃.csv
 **Description:** **Characterisation of glucokinase from** **glucokinase
 from** ***Escherichia coli*** **(EcGK) wild type (WT)** ##### Variables *
 Time (min):0-60 * Log of residual activity:0-2 * SD:The values represent
 mean values ± standard deviation of three independent experiments (n = 3).
 #### File: Fig2a_specific_activity_PsGK_WT.csv
 **Description:** **Characterisation of glucokinase
 from*Pseudoalteromonas*** **sp. AS-131 (PsGK) wild type (WT)** #####
 Variables * Temp:0-80 * U/mg:0-80 * SD:The values represent mean values ±
 standard deviation of three independent experiments (n = 3). #### File:
 Fig2b_residual_activity_PsGK_WT_55℃.csv
 **Description:** **Characterisation of glucokinase
 from*Pseudoalteromonas*** **sp. AS-131 (PsGK) wild type (WT)** #####
 Variables * Time (min):0-60 * Log of residual activity:0-2 * SD:The values
 represent mean values ± standard deviation of three independent
 experiments (n = 3). #### File: Fig2b_residual_activity_PsGK_WT_60℃.csv
 **Description:** **Characterisation of glucokinase
 from*Pseudoalteromonas*** **sp. AS-131 (PsGK) wild type (WT)** #####
 Variables * Time (min):0-60 * Log of residual activity :0-2 * SD:The
 values represent mean values ± standard deviation of three independent
 experiments (n = 3). #### File: Fig2b_residual_activity_PsGK_WT_65℃.csv
 **Description:** **Characterisation of glucokinase
 from*Pseudoalteromonas*** **sp. AS-131 (PsGK) wild type (WT)** #####
 Variables * Time (min):0-60 * Log of residual activity:0-2 * SD:The values
 represent mean values ± standard deviation of three independent
 experiments (n = 3). #### File: Fig2b_residual_activity_PsGK_WT_70℃.csv
 **Description:** **Characterisation of glucokinase
 from*Pseudoalteromonas*** **sp. AS-131 (PsGK) wild type (WT)** #####
 Variables * Time (min):0-60 * Log of residual activity:0-2 * SD:The values
 represent mean values ± standard deviation of three independent
 experiments (n = 3). #### File: Fig2c_thermal_stability_EcGK_WT_exp.csv
 **Description:** **Characterisation of glucokinase from** **glucokinase
 from** ***Escherichia coli*** **(EcGK) wild type (WT)** ##### Variables *
 Temperature:5-70 * Fraction unfolded:-0.2-1.2 #### File:
 Fig2c_thermal_stability_EcGK_WT_fit.csv
 **Description:** **Characterisation of glucokinase from** **glucokinase
 from** ***Escherichia coli*** **(EcGK) wild type (WT)** ##### Variables *
 Temperature:0-70 * Fraction unfolded:0-1 #### File:
 Fig2c_thermal_stability_PsGK_WT_exp.csv
 **Description:** **Characterisation of glucokinase
 from*Pseudoalteromonas*** **sp. AS-131 (PsGK) wild type (WT)** #####
 Variables * Temperature:5-90 * Fraction unfolded:-0.2-1.2 #### File:
 Fig2c_thermal_stability_PsGK_WT_fit.csv
 **Description:** **Characterisation of glucokinase
 from*Pseudoalteromonas*** **sp. AS-131 (PsGK) wild type (WT)** #####
 Variables * Temperature:5-90 * Fraction unfolded:0-1 #### File:
 Fig3c_DTNB_EcGK_C20S.csv **Description:** **Time dependence of
 5’5’-dithiobis(2-nitrobenzonate) (DTNB) reaction for glucokinase from**
 ***Escherichia coli*** **(EcGK) C20S** ##### Variables * Reaction time
 (s):0-43250 * Number of cysteine:0-3 #### File: Fig3c_DTNB_EcGK_C60S.csv
 **Description:** **Time dependence of 5’5’-dithiobis(2-nitrobenzonate)
 (DTNB) reaction for glucokinase from** ***Escherichia coli*** **(EcGK)
 C60S** ##### Variables * Reaction time (s):0-43250 * Number of
 cysteine:0-3 #### File: Fig3c_DTNB_EcGK_C65S.csv **Description:** **Time
 dependence of 5’5’-dithiobis(2-nitrobenzonate) (DTNB) reaction for
 glucokinase from** ***Escherichia coli*** **(EcGK) C65S** ##### Variables
 * Reaction time (s):0-43250 * Number of cysteine:0-2 #### File:
 Fig3c_DTNB_EcGK_WT.csv **Description:** **Time dependence of
 5’5’-dithiobis(2-nitrobenzonate) (DTNB) reaction for glucokinase
 from*Escherichia coli*** **(EcGK) wild type (WT)** ##### Variables *
 Reaction time (s):0-43250 * Number of cysteine:0-4 #### File:
 Fig4_thermal_stability_PsGK_C325S_exp.csv **Description:** **Thermal
 melting curves of glucokinase from*Pseudoalteromonas*** **sp. AS-131
 (PsGK) C325S** ##### Variables * Temperature: 5-90 * Fraction
 unfolded:-0.2-1.2 #### File: Fig4_thermal_stability_PsGK_C325S_fit.csv
 **Description:** **Thermal melting curves of glucokinase
 from*Pseudoalteromonas*** **sp. AS-131 (PsGK) C325S** ##### Variables *
 Temperature:5-90 * Fraction unfolded:0-1 #### File:
 Fig4_thermal_stability_PsGK_WT_exp.csv **Description:** **Thermal melting
 curves of glucokinase from*Pseudoalteromonas*** **sp. AS-131 (PsGK) wild
 type (WT)** ##### Variables * Temperature:5-90 * Fraction
 unfolded:-0.2-1.2 #### File: Fig4_thermal_stability_PsGK_WT_fit.csv
 **Description:** **Thermal melting curves of glucokinase
 from*Pseudoalteromonas*** **sp. AS-131 (PsGK) wild type (WT)** #####
 Variables * Temperature:5-90 * Fraction unfolded:0-1 #### File:
 Fig5b_DTNB_EcGK_DS-H.csv **Description:** **Time dependence of
 5’5’-dithiobis(2-nitrobenzonate) (DTNB) reaction for glucokinase
 from** ***Escherichia coli*** **(EcGK)** **C20S C65S H312C (DS-H)** #####
 Variables * Reaction time (s):0-11100 * Number of cysteine:0-2 #### File:
 Fig5b_DTNB_EcGK_DS-L.csv **Description:** **Time dependence of
 5’5’-dithiobis(2-nitrobenzonate) (DTNB) reaction for glucokinase
 from*Escherichia coli*** **(EcGK)** **C20S C65S L313C (DS-L)** #####
 Variables * Reaction time (s):0-11100 * Number of cysteine:0-2 #### File:
 Fig5b_DTNB_EcGK_DS-S.csv **Description:** **Time dependence of
 5’5’-dithiobis(2-nitrobenzonate) (DTNB) reaction for glucokinase
 from*Escherichia coli*** **(EcGK)** **C20S C65S S309C (DS-S)** #####
 Variables * Reaction time (s):0-11100 * Number of cysteine:0-2 #### File:
 Fig5b_DTNB_EcGK_H312C.csv **Description:** **Time dependence of
 5’5’-dithiobis(2-nitrobenzonate) (DTNB) reaction for glucokinase
 from*Escherichia coli*** **(EcGK)** **H312C** ##### Variables * Reaction
 time (s):0-11100 * Number of cysteine:0-4 #### File:
 Fig5c_thermal_stability_EcGK_DS-H_exp.csv **Description:** **Thermal
 melting curves of glucokinase from *Escherichia coli*** **(EcGK)** **C20S
 C65S H312C (DS-H)** ##### Variables * Temperature:5-70 * Fraction
 unfolded:-0.2-1.2 #### File: Fig5c_thermal_stability_EcGK_DS-H_fit.csv
 **Description:** **Thermal melting curves of glucokinase from *Escherichia
 coli*** **(EcGK)** **C20S C65S H312C (DS-H)** ##### Variables *
 Temperature:5-70 * Fraction unfolded:0-1 #### File:
 Fig5c_thermal_stability_EcGK_DS-L_exp.csv **Description:** **Thermal
 melting curves of glucokinase from *Escherichia coli*** **(EcGK)** **C20S
 C65S L313C (DS-L)** ##### Variables * Temperature:5-70 * Fraction
 unfolded:-0.2-1.2 #### File: Fig5c_thermal_stability_EcGK_DS-L_fit.csv
 **Description:** **Thermal melting curves of glucokinase from *Escherichia
 coli*** **(EcGK)** **C20S C65S L313C (DS-L)** ##### Variables *
 Temperature:5-70 * Fraction unfolded:0-1 #### File:
 Fig5c_thermal_stability_EcGK_DS-S_exp.csv **Description:** **Thermal
 melting curves of glucokinase from *Escherichia coli*** **(EcGK)** **C20S
 C65S S309C (DS-S)** ##### Variables * Temperature:5-70 * Fraction
 unfolded:-0.2-1.2 #### File: Fig5c_thermal_stability_EcGK_DS-S_fit.csv
 **Description:** **Thermal melting curves of glucokinase from *Escherichia
 coli*** **(EcGK)** **C20S C65S S309C (DS-S)** ##### Variables *
 Temperature:5-70 * Fraction unfolded:0-1 #### File:
 FigS11_CD_EcGK_DS-H.csv **Description:** **Thermal melting curves of
 glucokinase from *Escherichia coli*** **(EcGK)** **C20S C65S H312C
 (DS-H)** ##### Variables * wavelength (nm):200-260 * Ellipticity
 (mdeg):-30-10 #### File: FigS11_CD_EcGK_DS-L.csv
 **Description:** **Thermal melting curves of glucokinase from *Escherichia
 coli*** **(EcGK)** **C20S C65S L313C (DS-L)** ##### Variables * wavelength
 (nm):200-260 * Ellipticity (mdeg):-30-10 #### File:
 FigS11_CD_EcGK_DS-S.csv **Description:** **Thermal melting curves of
 glucokinase from *Escherichia coli*** **(EcGK)** **C20S C65S S309C
 (DS-S)** ##### Variables * wavelength (nm):200-260 * Ellipticity
 (mdeg):-30-10 #### File: FigS11_CD_EcGK_WT.csv **Description:** **Thermal
 melting curves of glucokinase from *Escherichia coli*** **(EcGK)** **wild
 type (WT)** ##### Variables * wavelength (nm):200-260 * Ellipticity
 (mdeg):-30-10 #### File: FigS10b_SG-SG_DS-S.csv **Description:** **The
 distance between cysteine sulphur (SG) atoms glucokinase from**
 ***Escherichia coli*** **(EcGK) C20S C65S S309C (DS-S)** ##### Variables *
 Time (ns):0-50 * distance (Å):0-6 #### File: FigS10a_SG-SG_DS-L.csv
 **Description:** **The distance between cysteine sulphur (SG)
 atoms glucokinase from** ***Escherichia coli*** **(EcGK) C20S C65S L313C
 (DS-L)** ##### Variables * Time (ns):0-50 * distance (Å):0-8 #### File:
 FigS10c_SG-SG_DS-S.csv **Description:** **The distance between cysteine
 sulphur (SG) atoms glucokinase from** ***Escherichia coli*** **(EcGK) C20S
 C65S S309C (DS-S) containing pre-formed disulfide bond** ##### Variables *
 Time (ns):0-50 * distance (Å):0-5 #### File:
 FigS4_relative_activity_PsGK_WT.csv **Description:** **Plot of relative
 activity as a function of temperature of** ***Pseudoalteromonas*** **sp.
 AS-131 (PsGK) wild type (WT)** ##### Variables * Temp:0-80 * Relative
 activity:0-1 #### File: FigS5_Arrhenius_EcGK_WT.csv
 **Description:** **Arrhenius plot of the substrate turnover (*k*~cat~) of
 glucokinase from*Escherichia coli*** **(EcGK)** ##### Variables *
 1000/Temperature (K):3.2-3.65 * ln kcat:2-5 * SD:The values represent mean
 values ± standard deviation of three independent experiments (n = 3). ####
 File: FigS5_Arrhenius_PsGK_WT.csv **Description:** **Arrhenius plot of the
 substrate turnover (*k*~cat~) of glucokinase from*Pseudoalteromonas***
 **sp. AS-131 (PsGK)** ##### Variables * 1000/Temperature (K):3.2-3.65 * ln
 kcat:2-5 * SD:The values represent mean values ± standard deviation of
 three independent experiments (n = 3). #### File:
 FigS13_specific_activity_EcGK_DS-S.csv **Description:** **Plot of specific
 activity of glucokinase from*Escherichia coli*** **(EcGK) C20S C65S S309C
 (DS-S) as a function of temperature** ##### Variables * Temp:0-70 *
 U/mg:0-100 * SD:The values represent mean values ± standard deviation of
 three independent experiments (n = 3). #### File:
 FigS13_specific_activity_EcGK_WT.csv **Description:** **Plot of specific
 activity of glucokinase from*Escherichia coli***  **(EcGK) wild type (WT)
 as a function of temperature** ##### Variables * Temp:0-65 * U/mg:0-40 *
 SD:The values represent mean values ± standard deviation of three
 independent experiments (n = 3). #### File:
 FigS4_relative_activity_EcGK_WT.csv **Description:** **Plot of relative
 activity as a function of temperature of**  ***Escherichia
 coli*** **(EcGK) wild type (WT)** ##### Variables * Temp:0-65 * Relative
 activity:0-1 #### File: FigS6_residual_activity_PsGK_WT_70℃.csv
 **Description:** **Thermal stability of** **glucokinase
 from*Pseudoalteromonas*** **sp. AS-131 (PsGK) wild type (WT)** #####
 Variables * Time (min):0-60 * residual activity (%):0-100 * SD:The values
 represent mean values ± standard deviation of three independent
 experiments (n = 3). #### File: FigS6_residual_activity_EcGK_WT_45℃.csv
 **Description:** **Thermal stability of** **glucokinase from**
 ***Escherichia coli*** **(EcGK) wild type (WT)** ##### Variables * Time
 (min):0-60 * residual activity (%):0-100 * SD:The values represent mean
 values ± standard deviation of three independent experiments (n = 3). ####
 File: FigS6_residual_activity_EcGK_WT_50℃.csv **Description:** **Thermal
 stability of** **glucokinase from** ***Escherichia coli*** **(EcGK) wild
 type (WT)** ##### Variables * Time (min):0-60 * residual activity
 (%):0-100 * SD:The values represent mean values ± standard deviation of
 three independent experiments (n = 3). #### File:
 FigS6_residual_activity_EcGK_WT_55℃.csv **Description:** **Thermal
 stability of** **glucokinase from** ***Escherichia coli*** **(EcGK) wild
 type (WT)** ##### Variables * Time (min):0-60 * residual activity
 (%):0-100 * SD:The values represent mean values ± standard deviation of
 three independent experiments (n = 3). #### File:
 FigS6_residual_activity_PsGK_WT_65℃.csv **Description:** **Thermal
 stability of** **glucokinase from*Pseudoalteromonas*** **sp. AS-131 (PsGK)
 wild type (WT)** ##### Variables * Time (min):0-60 * residual activity
 (%):0-100 * SD:The values represent mean values ± standard deviation of
 three independent experiments (n = 3). #### File:
 FigS6_residual_activity_PsGK_WT_55℃.csv **Description:** **Thermal
 stability of** **glucokinase from*Pseudoalteromonas*** **sp. AS-131 (PsGK)
 wild type (WT)** ##### Variables * Time (min):0-60 * residual activity
 (%):0-100 * SD:The values represent mean values ± standard deviation of
 three independent experiments (n = 3). #### File:
 FigS6_residual_activity_PsGK_WT_60℃.csv **Description:** **Thermal
 stability of** **glucokinase from*Pseudoalteromonas*** **sp. AS-131 (PsGK)
 wild type (WT)** ##### Variables * Time (min):0-60 * residual activity
 (%):0-100 * SD:The values represent mean values ± standard deviation of
 three independent experiments (n = 3). #### File:
 FigS7a_residual_activity_PsGK_WT.csv **Description:** **Thermal stability
 of** **glucokinase from*Pseudoalteromonas*** **sp. AS-131 (PsGK) wild type
 (WT)** ##### Variables * Temp:0-80 * Residual activity (%):0-120 * SD:The
 values represent mean values ± standard deviation of three independent
 experiments (n = 3). #### File: FigS7b_residual_activity_PsGK_WT_exp.csv
 **Description:** **Thermal stability of** **glucokinase
 from*Pseudoalteromonas*** **sp. AS-131 (PsGK) wild type (WT)** #####
 Variables * Temp:0-80 * Residual activity (%):0-100 * SD:The values
 represent mean values ± standard deviation of three independent
 experiments (n = 3). #### File: FigS7b_residual_activity_PsGK_WT_fit.csv
 **Description:** **Thermal stability of** **glucokinase
 from*Pseudoalteromonas*** **sp. AS-131 (PsGK) wild type (WT)** #####
 Variables * Temp:0-80 * Residual activity (%):0-100 #### File:
 FigS7c_residual_activity_EcGK_WT_exp.csv **Description:** **Thermal
 stability of** **glucokinase from** ***Escherichia coli*** **(EcGK) wild
 type (WT)** ##### Variables * Temp:0-60 * Residual activity (%):0-100 *
 SD:The values represent mean values ± standard deviation of three
 independent experiments (n = 3). #### File:
 FigS7c_residual_activity_EcGK_WT_fit.csv **Description:** **Thermal
 stability of** **glucokinase from** ***Escherichia coli*** **(EcGK) wild
 type (WT)** ##### Variables * Temp:0-60 * Residual activity (%):0-100 ####
 File: FigS7a_residual_activity_EcGK_WT.csv **Description:** **Thermal
 stability of** **glucokinase from** ***Escherichia coli*** **(EcGK) wild
 type (WT)** ##### Variables * Temp:0-60 * Residual activity (%):0-120 *
 SD:The values represent mean values ± standard deviation of three
 independent experiments (n = 3). #### File:
 FigS9_relative_activity_PsGK_C325S.csv **Description:** **Relative
 activity of** **glucokinase from*Pseudoalteromonas*** **sp. AS-131 (PsGK)
 C325S** ##### Variables * Temp:0-80 * relative activity:0-1 #### File:
 FigS9_relative_activity_PsGK_WT.csv **Description:** **Relative activity
 of** **glucokinase from*Pseudoalteromonas*** **sp. AS-131 (PsGK) wild type
 (WT)** ##### Variables * Temp:0-80 * relative activity:0-1 #### File:
 FigS9_specific_activity_PsGK_C325S.csv **Description:** **Specific
 activity of** **glucokinase from*Pseudoalteromonas*** **sp. AS-131 (PsGK)
 C325S** ##### Variables * Temp:0-80 * U/mg:0-140 * SD:The values represent
 mean values ± standard deviation of three independent experiments (n = 3).
 #### File: FigS9_specific_activity_PsGK_WT.csv **Description:** **Specific
 activity of** **glucokinase from*Pseudoalteromonas*** **sp. AS-131 (PsGK)
 wild type (WT)** ##### Variables * Temp:0-80 * U/mg:0-80 * SD:The values
 represent mean values ± standard deviation of three independent
 experiments (n = 3). #### File: Fig3d_DTNB_PsGK_C156S.csv
 **Description:** **Time dependence of 5’5’-dithiobis(2-nitrobenzonate)
 (DTNB) reaction for glucokinase from*Pseudoalteromonas*** **sp. AS-131
 (PsGK) C156S** ##### Variables * Reaction time (s):0-43250 * Number of
 cysteine:0-1 #### File: Fig5b_DTNB_EcGK_WT.csv **Description:** **Time
 dependence of 5’5’-dithiobis(2-nitrobenzonate) (DTNB) reaction for
 glucokinase from *Escherichia coli*** **(EcGK) wild type (WT)** #####
 Variables * Reaction time (s):0-11100 * Number of cysteine:0-4 #### File:
 Fig3d_DTNB_PsGK_WT.csv **Description:** **Time dependence of
 5’5’-dithiobis(2-nitrobenzonate) (DTNB) reaction for glucokinase
 from*Pseudoalteromonas*** **sp. AS-131 (PsGK) wild type (WT)** #####
 Variables * Reaction time (s):0-43250 * Number of cysteine:0-2
 **Supplemental information uploaded to Zenodo:** 
 PsGK_SI_revised_clean.docs contains the supplementary tables and figures
 with respective legends.