Contribution of a C-Terminal Extension to the Substrate Affinity and Oligomeric Stability of Aldehyde Dehydrogenase from Thermus thermophilus HB27

Aldehyde dehydrogenase enzymes (ALDHs) are widely studied for their roles in disease propagation and cell metabolism. Their use in biocatalysis applications, for the conversion of aldehydes to carboxylic acids, has also been recognized. Understanding the structural features and functions of both prokaryotic and eukaryotic ALDHs is key to uncovering novel applications of the enzyme and probing its role in disease propagation. The thermostable enzyme ALDHTt originating fromThermus thermophilus, strain HB27, possesses a unique extension of its C-terminus, which has been evolutionarily excluded from mesophilic counterparts and other thermophilic enzymes in the same genus. In this work, the thermophilic adaptation is studied by the expression and optimized purification of mutant ALDHTt-508, with a 22-amino acid truncation of the C-terminus. The mutant shows increased activity throughout production compared to native ALDHTt, indicating an opening of the active site upon C-terminus truncation and giving rationale into the evolutionary exclusion of the C-terminal extension from similar thermophilic and mesophilic ALDH proteins. Additionally, the C-terminus is shown to play a role in controlling substrate specificity of native ALDH, particularly in excluding catalysis of certain large and certain aromatic ortho-substituted aldehydes, as well as modulating the protein’s pH tolerance by increasing surface charge. Dynamic light scattering and size-exclusion HPLC methods are used to show the role of the C-terminus in ALDHTt oligomeric stability at the cost of catalytic efficiency. Studying the aggregation rate of ALDHTt with and without a C-terminal extension leads to the conclusion that ALDHTt follows a monomolecular reaction aggregation mechanism.


■ INTRODUCTION
Thermophiles are bacteria that are abundant in versatility, living in extreme temperature environments such as volcanoes, hot springs, and deep-sea thermal vents.Proteins expressed by thermophilic bacteria have been applied in industry and research, from food production and detergents to genetic engineering applications. 1The mechanisms by which these proteins withstand high temperatures, often exceeding 100 °C, are less well-known.The thermodynamic equilibria between the folded and denatured states are kept in balance by a range of evolutionary adaptations including, but not limited to, increased hydrophobicity of residues and resulting secondary structures, increased number of disulfide bonds, and formation of salt bridges between residues. 2Additionally, thermophilic enzymes tend to form larger oligomeric states, indicating the role of quaternary structure in their stability. 3Due to a multitude of mechanisms by which these proteins survive, it is often not possible to identify specific structural features that can be studied in depth.
C-Terminus adaptations found in some thermophilic proteins have been recognized as such structural features, or stability "tags", as they have a major impact on overall protein thermostability and oligomeric state.A molecular modeling study on an RNase H1 enzyme from the hyperthermophile Sulfolobus tokodaii suggests that the extension of the Cterminus in the enzyme plays the sole role of stabilization by anchoring a range of disulfide bridges through the protein core. 4Thermally stabilizing C-terminal tails have also been discovered in the Thermus genus, where they may act as "clamps", by a network of hydrophobic interactions on the surface of the cytochrome c 552 protein. 5A range of 1,4-αglucan-branching enzymes (GBEs) studied from the bacteria Geobacillus thermoglucosidans (optimal growth ∼60 °C) revealed a unique 26-amino acid extension of the C-terminus which is required for structural stability. 6A recent study of thermophilic DNA helicases show increased target binding due to an N-and C-termini extension, in comparison with nonthermophilic counterparts. 7C-Termini extensions have also been identified in Baker's yeast (Saccharomyces cerevisiae), with a role in mitigating aggregation and deactivation of the structure. 8In particular, C-terminal tails with a helical conformation have been demonstrated as oligomeric stabilizers by increasing interactions at the surface of the molecule. 9α-Helical C-termini have also been propositioned to increase the rigidity of thermophilic proteases. 10−13 For example, a fungal xylanase was found to possess a C-terminal extension, which decreased its catalytic efficiency by approximately 2.4 times. 13The catalytic activity of human ubiquitin-specific protease (USP) enzyme (USP7) has been shown to be tightly controlled by a 19-amino acid C-terminal extension, blocking excessive substrate binding. 14n ALDH enzyme originating from a Thermus thermophilus bacterium (ALDHTt, UniProtKB Q72KD3), which possesses a structurally unique C-terminal extension, has recently been discovered, during crystallization of a caa 3 -cytochrome c oxidase. 15The feature was deemed unique as it resembled the C-terminus extensions of dimeric ALDHs; however, its position over the catalytic tunnel linked it with the opposing monomer, forming a D2 or "222" dihedral point group symmetry.Additionally, the closely related T. thermophilus HB8 aldehyde dehydrogenase protein possesses a significantly shorter tail, with a full peptide length of 515 amino acids as opposed to 530 for ALDHTt, yet retains its thermophilic properties.Alignment of the C-terminal tails of various ALDH proteins can be found in Figure 1.The C-terminal extension of ALDHTt was recognized for its role in the protein's stability by the creation of two mutants, ALDHTt-515 and ALDHTt-511 (PDB entry: 6FKU and 6FKV, respectively).The sequence of ALDHTt-511 was deposited as ALDHTt-508; however, further examination of the sequence by MALDI-TOF in this manuscript has revealed three additional amino acids, AQM, not truncated from the C-terminus.The FASTA sequence on PDB was wrongly deposited during this previous study, and we present the correct sequence for ALDHTt-508 in Figure S1.
The C-terminal tail of ALDHTt consists of 30 amino acids (see Figure 1), encompassing a three-turn helix, made up of the last six residues in the sequence.Prolines residing at positions P518, P521, and P523 prevent the formation of secondary structure across the substrate access tunnel over which the extension is positioned. 16In Figure 2D, the relation between the substrate access tunnel and the unique extension of the tail is depicted, which is governed by the side chain of lysine at position 105 of the opposing monomer.The Cterminal tail in related ALDH proteins may be divided into  three loops, or turns, termed the "notch", the "crook" and the "hook", respectively.Most dimeric ALDHs C-terminal tails end at the "notch", which lays between the residues Ser503 and Gly504 in ALDHTt.The C-terminal tail in ALDHTt however continues toward the N-terminus of the corresponding symmetrical monomer.Interestingly, the C-termini of aldehyde dehydrogenases produced by other closely related thermophilic bacterial strains are conserved until residue 515 (see Figure 1).Subsequent amino acids do not show any level of homology among the species.The "crook" is positioned directly over the substrate tunnel and governed by the interaction of Leu508 and Asp512.The residue Gln510 forms a hydrogen bond with an alanine, Ala465, located in the substrate anchoring region within the tunnel (Figure 2A).Through this strong, noncovalent interaction, the C-terminal tail of the ALDHTt plays an important role in enzyme activity, unlike most other ALDH proteins where the access site is open, or the tail is repositioned during catalysis. 17he ALDH superfamily of enzymes have only recently been recognized for their application in biocatalysis, having the potential to replace chemical methods in the conversion of aldehydes to carboxylic acids, particularly due to their safety, ease of use, and minimal environmental impact. 18Exploration of the structure and function of ALDHs is vital to uncovering their potential applications in this area.Additionally, the presence of C-terminal tails has interesting implications for the structural and thermal stability of proteins and warrants further investigation.In this paper, a mutant of ALDHTt-native is created with a fully open substrate tunnel by removal of the key 22 amino acids and 3-turn helix of the C-terminal extension, termed ALDHTt-508.The effect on function and stability of the truncation is explored by screening of pH and temperature profiles, substrate specificity, size exclusion highperformance liquid chromatography (SE-HPLC), and dynamic light scattering (DLS).The data presented in this manuscript suggests that the C-terminus not only has a vital role in stabilization of the tetrameric structure but also plays a role in activity control.The characterizations described herein aid in understanding the effect of the C-terminus extension in bacterial thermophiles and possibly offer a new avenue to explore for protein stabilization.
Methods.Transformation, Expression, and Purification of ALDHTt-508.PET22b(+) plasmids containing the sequence gene for ALDHTt-native, 530 amino acids, and ALDHTt-508 were constructed, as previously described elsewhere. 15The plasmids were stored at −20 °C.Escherichia coli (BL21)DE3 cells were used for the recombinant production of both proteins.The construct containing the gene encoding for ALDHTt-native and ALDHTt-508 with a shortened Cterminal tail was transformed into chemically competent E. coli BL21 (DE3) cells using heat shock and incubation in S.O.C (super optimal broth with catabolite repression) media.
After an overnight culture in LB media, the cells were incubated in ZY autoinduction media, as previously described elsewhere. 19The culture was grown for 48 h at 25 °C under constant shaking at 200 rpm.The cells were collected by centrifugation at 6000 × g for 15 min in a 4 °C precooled rotor centrifuge.The cell pellet was resuspended in a Lysis buffer (Tris 20 mM, 2-mercaptoethanol 5 mM, imidazole 10 mM, NaCl 500 mM), 2 mg/mL DNase, 25 mg/mL lysozyme, and 1 M MgCl 2 ).The solution was mixed gently on ice using a pipet and transferred to a −80 °C freezer for a minimum of 12 h.
Purification of ALDHTt-508 was conducted by nickel affinity chromatography using a chelating Sepharose fast flow resin (Cytiva Life Sciences) charged with Ni 2+ ions.Intracellular proteins were separated from lysate by centrifugation, heat-shocked at 65 °C for 5 min to remove unstable intracellular contaminants, and filtered using a 0.45 μm nylon filter.The protein was eluted from affinity chromatography by FPLC (A ̈KTAprime plus, Cytiva Life Sciences) using an imidazole gradient.Absorbance was monitored at 280 nm.The protein was dialyzed overnight using cellulose dialysis tubing, MWCO 8000 Da (Fisher Scientific), to remove imidazole from the storage buffer.The protein was snapfrozen and stored at −80 °C for further analysis.Size exclusion chromatography was conducted to evaluate the effect of lysate heat-shock on the protein using a pre-equilibrated HiLoad 16/ 60 Superdex 200 prep grade column (GE Healthcare).The mobile phase used during equilibration and elution consisted of 50 mM Tris-HCl pH 7.5, 5 mM β-mercaptoethanol, and 150 mM NaCl.
ALDHTt-native was prepared as described elsewhere. 15The purity and presence of expressed protein was confirmed by 12% SDS-PAGE and Western blot, which utilized conjugation of Anti 6× His-peroxidase antibody to one-step TMB substrate solution (ThermoFisher Scientific).Quantitative purity was confirmed by SE-HPLC, by injecting 50 μL of a 0.5 mg/mL sample onto a SE-HPLC column (AdvanceBio SEC 300 Å, 2.7 μm, 8 × 300 mm, Agilent Technologies) at a flow rate of 1 mL/min.The purity of the samples was calculated by calculating the percentage of the native protein peak area to the total peak area.
MALDI-TOF.The samples were buffer exchanged into 0.1% trifluoroacetic acid (TFA) and concentrated to 3 mg/mL.Approximately 7.6 mg of 2,5-dihydroxyacetophenone (2,5DHAP) was dissolved in a 500 μL solution of 75:25 v/v ethanol, 18 mg/mL diammonium hydrogen citrate in water to make up the matrix solution.A 1 μL sample solution was mixed with 1 μL of 2% TFA solution and 1 μL of matrix solution.A 1 μL drop was deposited on a Bruker ground steel target plate and allowed to dry at room temperature.Intact mass spectra were obtained in a Bruker UltrafleXtreme MALDI TOF/TOF device equipped with a SmartBeam2 laser in linear positive mode with an acceleration gain of 20 kV, 500 ns ion extraction delay, and 2750 V detector gain.Data was acquired and processed using the Compass 1.4 software suit from Bruker.Peak masses were assigned using the Centroid algorithm in FlexAnalysis, and data were externally calibrated using the Protein Standard II from Bruker.
pH and Temperature Activity Parameter Screening.The pH and temperature stability of ALDHTt-508 was tested using a range of temperatures and pH, previously described for ALDHTt for the evaluation of the impact of the mutation on Biochemistry the pH and temperature activity profile of the protein. 18A temperature range of 20−50 °C and pH range of 2−10 were analyzed for NAD+ dependent activity, using the substrate hexanal.Temperatures above 50 °C were not analyzed due to the volatility of hexanal.pH ranges of 2−10 were achieved using a range of 10 mM citrate buffers (2−5), 10 mM potassium phosphate buffers (6−8), and 10 mM Tris-HCl buffers (9−10).
NAD+ Coupled Enzymatic Assay.The enzyme assays for both ALDHTt-native and ALDHTt-508 were conducted using 60 nM ALDH, 0.4 mM NAD+ (Nicotinamide adenine dinucleotide sodium salt, Sigma-Aldrich), and 1 mM of the substrate hexanal (Sigma-Aldrich) in a volume of 1 mL.All solutions were made in 10 mM potassium phosphate buffer (pH 8.0), and the rise in absorbance was measured every 5 s for 120 s.All enzyme assays were conducted in triplicate.The enzyme activity was calculated by the change in NADH concentration in the solution per minute and expressed in 1 U/ mg (specific enzyme units), where 1 U = 1 μmol of NADH/ min.All enzymatic assays were performed using an Evolution 201 Series UV−vis spectrophotometer (ThermoFisher).Kinetic parameters were evaluated using GraphPad Prism 8.0.1.
Substrate Screening.A range of aldehydes were screened to observe the effect of the loss of the C-terminal extension on ALDHTt substrate specificity and affinity.The experiments were conducted at room temperature (25 °C) and at the optimum operating temperature of the enzyme (50 °C).Approximately 2 mM of the following aromatic and aliphatic aldehydes were used in the experiments: cyclo-hexanecarboxaldehyde, furfural, acetaldehyde, benzaldehyde, trans-cinnamaldehyde, o-tolualdehyde, p-tolualdehyde, propanal, and citral.
Oligomeric Stability Testing.SE-HPLC was conducted on heat-treated ALDHTt-native and ALDHTt-508 samples to probe the oligomeric stability of the proteins.All samples were injected at room temperature onto a SE-HPLC column (AdvanceBio SEC 300 Å, 2.7 μm, 8 × 300 mm, Agilent Technologies) at a flow rate of 1 mL/min using an Agilent 1260 Infinity Series HPLC system (Agilent Technologies).The signals were collected by UV absorption at 220 and 280 nm.
The samples were adjusted to 0.5 mg/mL in 10 mM potassium phosphate buffer and incubated on a block heater at 65 °C for 5, 10, 15, and 20 min.The samples were then centrifuged at 10000 × g for 10 min to remove large particles.The column was calibrated with the following mixture: Thyroglobulin bovine MW ∼ 670 kDa, γ-globulins from bovine blood with MW ≈ 150 kDa, ovalbumin with MW ≈ 44.3 kDa, ribonuclease A type I-A with MW ≈ 13.7 kDa, and p-aminobenzoic acid (pABA) with MW ≈ 137 Da.Approximately 50 μL of each sample was injected into the column, which was pre-equilibrated with 10 mM potassium phosphate buffer.The molecular masses of the proteins and their oligomeric states were determined from the calibration plot of the standards.
Dynamic Light Scattering (DLS).The steady-state hydrodynamic diameter (d H ) of proteins ALDHTt-native and ALDHTt-508 was measured using dynamic light scattering (DLS) on a Zetasizer Nano-ZSP (Malvern Instruments, UK) at 25 °C in three independent experiments.The samples were prepared by dilution or buffer exchange into 10 mM potassium phosphate buffer at a concentration of 800 μg/mL and filter-sterilized by a 0.22 μm PTFE filter to remove large aggregates.The intensity of scattered light was measured at 173°with an avalanche photodiode.The instrument was fitted with a 633 nm He−Ne laser.The hydrodynamic diameter was calculated from the Stokes−Einstein equation: 20 where D H is the hydrodynamic radius, T is absolute temperature (varied), η s is the solvent viscosity (10 mM potassium phosphate buffer was used as solvent), and k B is the Boltzmann constant.The measurements were taken at a scattering angle of 90°to reduce scattering from dust in the solution.The temperature of aggregation (T agg ) was determined using multiparameter analysis (monitoring both size and intensity of scattered light as a function of temperature).The temperature was increased from 30 to 70 °C with a 0.5 °C step increment and a 60 s equilibration time of the sample at each temperature step.The T agg was determined by fitting the experimental data to a plateau followed by a one-phase association model (eq 2) using Prism software (version 10.0.1).

I e k T T ( lim
) 1 ( ( )) where T 0 is the time at which an increase in intensity is observed (T agg ), lim I is the maximum intensity reached during the experiment, and k I is the kinetic rate constant of aggregation.
All measurements were conducted in a minimum of two independent experiments.
Thermal aggregation was measured by measuring the scattering intensity and D H of the sample as a function of time at the respective T agg of each protein, 65 and 80 °C.The aggregation kinetics were fit to an exponential growth curve using Prism software (ver 10.0.1).
Circular Dichroism (CD).Untreated secondary structure spectra of ALDHTt-native and ALDHTt-508 were analyzed using a Chirascan Plus CD spectrophotometer (Applied PhotoPhysics Ltd., UK).The spectra were recorded in the far-UV wavelength range 180−250 nm.The path length was 1 cm with a wavelength step of 1 nm.Wavelength scans were obtained in triplicate of protein solutions of 0.005 mg/mL in 10 mM potassium phosphate buffer, pH 8.0, the spectra of which were also used as a blank.A quartz cuvette was used for the analysis.All spectra were reported in CD units (mdeg).The spectra were averaged, background corrected using the blank, and allowed 6-point Savitsky−Golay smoothing using the accompanying software Pro-Data Chirascan.Secondary structure composition of both proteins was estimated using the K2D2 predictor tool available online at dichroweb.cryst.bbk.ac.uk. 21RESULTS

Recombinant Production and Purification of the Kinetically Active Mutant ALDHTt-508 and Assessment
of Lysate Heat-Treatment.The overexpression of ALDHTt-508 in E. coli cytosol was confirmed by a strong band present in the lysate soluble fraction made up of E. coli intracellular proteins at approximately 57−58 kDa via SDS-PAGE (Figure 3A, lane 1) and confirmed to contain the His-tagged mutant by Western blot (Figure 3B, lane 1).The purity of samples after

Biochemistry
Ni-affinity was confirmed to be 89.9 ± 0.2%, as calculated by ratio of peak area of protein to contaminants by SE-HPLC (see Table 1 below) and qualitatively by 12% SDS-PAGE (Figure 3A, lane 3).The protein was seen to fully bind to the resin as little to no protein is visualized in the nickel-affinity flowthrough (Figure 3A, lane 2).The protein of interest eluted similarly to native ALDHTt, at 200 mM imidazole (Figures S2  and S3).Most contaminating E. coli proteins are seen eluted between 0 and 50 mM imidazole, as per Figure S2.Samples subjected to SEC confirm the purity and structural integrity of the protein after affinity chromatography by a singular peak at an 80 mL elution volume (Figure S3B).The structural integrity of the tetramer was retained under varied levels of lysate heat shock (0−15 min), as confirmed by a singular peak at 80 mL elution volume during gel filtration (Figure S7); however, the yield was majorly affected by the lack or excess of heat treatment of the lysate (Table 1).
Table 1 shows the details of the effect of various purification approaches used.Each 1 L of ZY-auto induction culture purified by a single affinity chromatography step yielded on average 22.6 ± 8.6 mg/L, when the cell lysate was treated to 5 min of heat shock.The yields were approximately 45% lower than those obtained from the production of ALDHTt-native (66.5 ± 10.5 mg/L).The protein, which was treated to only 5 min of heat treatment, was relatively homogeneous after Ni-affinity, achieving 89.9% purity.Treatment of the lysate to a 15 min heat shock step, as described elsewhere for production of native ALDHTt, 15,18 drastically lowers the specific activity of ALDHTt-508 after purification by Ni affinity and gel filtration.This technique is common for purification of thermozymes expressed in mesophilic hosts. 22The truncated enzyme displays a specific activity of 0.453 ± 0.041 U/mg when purified to homogeneity by gel filtration after a 15 min heat shock step.The enzyme activity of ALDHTt-508 increases to 0.953 ± 0.035 U/mg when this step is reduced to 5 min.Complete omittance of this step does not improve the retention of enzymatic activity, with activity reaching 0.545 ± 0.037 U/mg.Furthermore, treatment of ALDHTt-508 to a 15 min heat shock step results in a deterioration of the protein, resulting in two distinct peaks detectable in SE-HPLC (Figure S8).The major peak present in heat-treated lysate samples was the native tetramer (27.02% of total peak area); however, the second biggest peak (6.65% of total peak area) corresponded to the molecular weight of an ALDHTt-508 hexamer (MW ≈ 380 kDa).Other peaks corresponded to trimeric (∼190 kDa) and dimeric (118 kDa) species.
The catalytic activity is improved for ALDHTt-508 when the heat shock purification step is optimized.Previously, the production of ALDHTt-native has been optimized to include a 15 min heat shock step, with reduction of this purification step As detectable by the described SE-HPLC method at 280 nm.b Represents percentage of fully folded protein corresponding to the tetrameric peak of ALDHTt-508.

Biochemistry
resulting in a 40% loss of yield. 15,18When both proteins are treated to a 15 min heat shock step, the activity of ALDHTt-508 drops to ∼70% of that achievable by ALDHTt-native, which prompted optimization of this step in production of ALDHTt-508.Reduction of the lysate heat treatment time of ALDHTt-508 improved yields by 58% and improved the enzymatic activity by ∼50% (see Table 1).Indeed, affinity purification of ALDHTt-native under 5 min lysate heat treatment resulted in a specific activity of 0.542 ± 0.011 U/ mg, slightly lower than that of ALDHTt-508 treated to the same purification steps (0.564 ± 0.120, see Table 1).The purity of ALDHTt-508 is marginally improved by a second gel filtration step, increasing from 82−90 to 96−100% (Table 1).There is no significant difference in purity of the protein when treated to 0 or 5 min of heat treatment, and therefore, the step does not serve any beneficial function in the process.Retention of the 15 min heat treatment had a profound impact on purity, due to the presence of nonspecific oligomer states (aggregates) and cleaved protein states.The cleavage of ALDHTt-508 at increased temperatures is not related to the 6xHis-tag, as suspected, as no irregular elutions were noticed during nickel affinity purification.
Truncation of the C-Terminus Broadens the Substrate Specificity of ALDHTt.The presence and molecular mass of the protein was confirmed by MALDI-TOF (Figure S4).The molecular mass of the 508 amino acid protein was 57055.97Da, as measured by MALDI-TOF, comparable to the theoretical molecular mass returned by the Expasy Molecular Weight Analysis Tool, 57079.02Da (https://web.expasy.org,accessed 26th of April, 2023).This confirmed the successful expression of the mutant protein.
Due to the position of the C-terminal tail over the substrate access tunnel in the oligomeric state and the apparent impact on the catalytic rate of the protein, a detailed pH and temperature profile was conducted.The pI of ALDHTt-508 remained largely unchanged upon the mutation (pI ALDHTt-508 = 6.53, pI ALDHTt-native = 6.40), and therefore, no major change was observed for the optimum working pH of the protein (Figure 4B).The temperature profile of ALDHTt-508 is largely similar to that of ALDHTt-native, with a gradual increase to 0.952 ± 0.032 U/mg specific enzyme activity at 50 °C, showcasing the mutant's suitability to high-temperature catalytic applications (Figure 4A).The mutant protein displayed higher rates of activity at temperatures of 30, 40, and 50 °C, albeit the truncation did not affect activity at lower temperature.Similarly, the pH profile resembles the pH profile of ALDHTt-native, with an optimum operating pH of 8.0 (1.074 ± 0.031 U/mg).ALDHTt-508 was active at pH 3− 10, while ALDHTt-native showed activity only in the pH range of 5−10.The mutant protein is more active than ALDHTtnative at all pHs tested. 18ptimum catalytic conditions for the enzyme, using hexanal as a substrate, were found to be 50 °C at pH 8.0.As native ALDHTt follows Michaelis−Menten kinetics, values for K cat, V max , and K m were calculated for the ALDHTt-508 protein at the experimentally derived optimum pH and temperature and at 25 °C (room temperature).Results clearly indicate the thermophilic application of ALDHTt-508, with a 3.6-fold increase in the rate of reaction (V max ) from 25 to 50 °C and a proportional increase in the amount of NAD+ reduced per second (K cat ) of 3.8-fold (Table 2, Figure S5).Additionally, the enzyme reaches maximum velocity at much lower concentrations of substrate at 50 °C, indicated by the decrease in the halfway point of the reaction, K m , from 1.0078 ± 0.6021 mM at 25 °C to 0.1040 ± 0.0093 mM at 50 °C.The kinetic activity of ALDHTt-508 using the cofactor NADP+ was also calculated at 50 °C using hexanal.The enzyme was significantly less active using the larger cofactor, with a specific activity of 0.1915 ± 0.0341 U/mg (Figure S6, Table 2).When compared with an ALDHTt-native control, ALDHTt-508 showed more robust kinetic parameters using NAD+ as a cofactor at both mesophilic and thermophilic temperatures.A minor increase in  The final volume of the assay was 1 mL.

Biochemistry
the activity of the truncated enzyme was also observed when the larger cofactor, NADP+, was used in the assay.ALDHTt-native has recently been reported as having a wide substrate specificity toward both aromatic and aliphatic aldehydes. 18To observe if the truncation of the C-terminus influenced substrate specificity, a range of aliphatic and aromatic aldehydes were screened at 25 and 50 °C, pH 8.0 (Figure 5). Figure 5B shows that the truncated mutant, ALDHTt-508, is capable of oxidizing both aliphatic (hexanal, propanal, acetaldehyde, and citral), aromatic (benzaldehyde, ptolualdehyde, o-tolualdehyde, and furfural), and cyclic (cylohexaneboroaldehyde) compounds at a thermophilic temperature of 50 °C.The mutation at the C-terminus alters the specificity and activity of the enzyme toward the tested aldehydes.Removal of the C-terminus causes a total loss of enzymatic activity toward the aromatic compound transcinnamaldehyde at mesophilic temperatures (Figure 5).The enzyme retains a similar, minimal, affinity toward benzaldehyde at both 50 and 25 °C; however, it gains a substantial affinity toward propanal, and to a lesser extent, citral and furfural.At 50 °C, ALDHTt-508 shows a greater activity toward all of the aldehyde substrates tested.ALDHTt-508 also possesses a broader substrate scope than ALDHTt at 50 °C operating temperature in pH 8.0, capable of oxidizing o-tolualdehyde, citral, and furfural.The increase in specific enzymatic activity for the cyclic compound, cyclohexanecarboxaldehyde, is 3-fold, while affinity toward the aliphatic propanal increases by 6-fold.Unlike ALDHTt-native, ALDHTt-508 displays a higher activity toward propanal (0.238 ± 0.022 U/mg) than hexanal (0.172 ± 0.027 U/mg) at room temperature.The truncated enzyme is significantly more active toward the aliphatic aldehydes hexanal and propanal at room temperature than ALDHTt-native.A plot of the relative activity of each protein toward each substrate tested, with respect to the model substrate, hexanal, can be found in the Supporting Information (Figure S7).
Influence of the C-Terminal Tail on the Oligomeric and Thermal Stability of ALDHTt.Oligomeric dissociation and aggregation during thermal stress to both proteins, with and without a C-terminal extension, was examined using SE-HPLC and DLS.Determination of the oligomeric forms present in purified solutions of ALDHTt-native and ALDHTt-508 showed relative instability of ALDHTt-508 in solution, and a tendency toward monomeric (2.50%) and dimeric (1.71%) oligomeric states (see Table 3).There was also an  increment of aggregates in solution for ALDHTt-508, increasing from 1.15% for ALDHTt-native to 5.92% for ALDHTt-508 in 10 mM potassium phosphate buffer, pH 8.0.Incubation of the protein solutions at 65 °C led to a gradual loss of tetrameric structure for the native protein ALDHTtnative, decreasing from 98.8 to 93% over a period of 20 min, with a fraction shifting into soluble aggregates.SE-HPLC of ALDHTt-508 was not representative of the behavior of the protein in solution.For samples heated from 5 to 15 min at 65 °C, there is a decrease in the number of soluble aggregates in solution; however, extensive precipitation of the protein, which was filtered prior to injection, contradicted this result.The decrease in monomeric and dimeric states upon heating suggests that the structural fragments are involved in the formation of large insoluble aggregates.The hydrodynamic diameter, as determined by DLS, displayed values of 11.44 ± 0.24 nm for the native protein and 14.40 ± 2.37 nm for ALDHTt-508, which is in accordance with the diameter reported for other globular proteins of similar molecular weight and with the predicted molecular weight using the Zetasizer Nano-ZSP instrument (264 and 212 kDa, respectively). 23The values of experimentally derived D H reported are a weighted average obtained from the Zetasizer Nano-ZSP instrument, which creates a bias toward larger (aggregate) populations. 24he higher hydrodynamic diameter reported for ALDHTt-508 is therefore likely to be caused by the higher presence of aggregates in the sample (5.9% as measured by SE-HPLC) which were not filtered (D H < 0.22 μm).The size distribution by intensity of ALDHTt-508 shows three main populations present, at 11.14 ± 0.43 nm, 176.62 ± 86.39 nm, and >2 μm, with peak 1 corresponding to ∼90% of the total distribution area (Figure S9).This indicates that the true D H of ALDHTt-508 corresponds to 11.14 ± 0.43 nm.Both the SE-HPLC and DLS results show a destabilization of the tetrameric structure of ALDH upon C-terminal removal and a tendency toward formation of monomeric and dimeric species in solution.
The stability of the oligomeric ALDHTt-native and its mutant, ALDHTt-508, was studied using dynamic light scattering (DLS).The aggregation profiles of both proteins as a function of the temperature are shown in Figure 6.There was a significant change in both the increasing hydrodynamic diameter (D H ) and the scattering light intensity (kcps) as a function of temperature between the two proteins.The onset of aggregation for ALDHTt-native was determined at 47 °C, 6 °C higher than that of ALDHTt-508 (41 °C).Additionally, heating of ALDHTt-508 to 70 °C resulted in an increase in the average D H to >2 μm due to the formation of precipitates, while the D H of the native protein increased to only 24 nm.This is reflected by the magnitude of scattering intensity measured by DLS (ALDHTt-native 70 °C = 4,525 kcps, ALDHTt-508 70 °C = 31,487 kcps, data not shown).
Aggregation kinetics of both ALDHTt-native and ALDHTt-508 was measured by DLS at 65 and 80 °C (Table 4, Figure 7).The aggregation mechanisms of both proteins followed an exponential model of association at the initial stages of aggregation.The increase in intensity for both proteins reached a plateau, and therefore can be defined by the following equation: )   where I lim is the limiting intensity [I] at t → ∞ and k 1 is the first-order rate constant.Neither of the proteins show evidence of intermediate formation upon thermal stress, either in SE-HPLC or DLS as suggested previously for a structurally similar ALDH from Pseudomonas aeruginosa (PaBADH). 25This leads us to the conclusion that ALDHTt follows a monomolecular aggregation mechanism with no creation of intermediate oligomeric species.At temperatures of 65 and 80 °C, the presence of the C-terminal tail in ALDHTt-native slows the rate of aggregation (k I ) by 3-fold and 2-fold, respectively.The presence of the C-terminal extension also prevents the formation of large insoluble aggregates at 65 °C.
For ALDHTt-508 at 65 and 80 °C, the ascending kinetics at the beginning of the experiment follow the same exponential model shown in eq 3, where the I lim value denoted the point at which all protein was involved in aggregate formation; however, there is a gradual descent of the curve past the point of I lim , suggesting the formation and sedimentation of precipitates (D H > 2000 nm), which are not detected using the DLS technique. 26,27At 80 °C, both proteins precipitate out of solution, with ALDHTt-native precipitating at ∼21 min, compared to 6 min for ALDHTt-508.ALDHTt-native possessed a lower value of I lim for both temperatures studied (2808.0 ± 674.2 kcps at 65°and 40,756.6 ± 15,136.6 kcps at 80°).
The dependence of hydrodynamic diameter on scattering intensity of ALDHTt-native and ALDHTt-508 during a temperature ramp experiment of 30−70 °C are shown in Figure 8.The relationship between the scattered light intensity and the hydrodynamic diameter is linear.The plots indicate that during heating of ALDHTt-508 to 70 °C, the size of the initial aggregates (i.e., D H at which the first increase in light scattering intensity is observed, D H , 0 ) is significantly larger than the size of the initial aggregates produced when heating native ALDHTt.The values of D H,0 for ALDHTt-508 and ALDHTt-native were determined graphically to be 23.34 and 11.71 nm, respectively.There is a much larger increase in the size of aggregates of ALDHTt-508 than that of ALDHTt-native during heating, with a complete conversion of the mutant protein into aggregates, which can be observed by the shift of the distribution of D H to a unimodal population at ∼200 nm (Figure S10).Toward higher temperatures, the D H of ALDHTt-508 reached a critical value and begins to drop, leading to the stochastic behavior seen in Figure 8 at D H > 400 nm. Figure 8. Dependence of hydrodynamic diameter on the intensity of the scattered light of ALDHTt-native (A) and The intensity and hydrodynamic diameter data was collected using the Zetasizer Nano-ZSP DLS and normalized using Prism GraphPad (Ver 10.0.1) and fit to a plateau, followed by a one-phase association equation.The mean of three independent experiments is presented.

Biochemistry
ALDHTt-508 (B).The vertical dotted line corresponds to the value of D H at room temperature.
The effect of the truncation on the surface behavior of ALDHTt was visualized by PyMOL (Figure 9).The threedimensional structure shows that the truncation exposes previously hidden residues that were involved in ionic bonding with the C-terminus, including Ala465, which interacts with Gln510 of the opposing monomer and holds the C-terminus across the aldehyde access tunnel of ALDHTt.The C-terminus can be seen in terminating in a nonpolar, previously described three-turn helix, which does not bond with any of the residues on the surface of the structure, but is however free to move, replicating the movement of previously described "caps" in ALDH active site regulation. 28The figure also shows exposure of the highly polar active site, increasing the flexibility of the molecule.
CD spectra representative of both the native ALDHTt protein and ALDHTt-508 are depicted in Figure S11.Both spectra were obtained in the Far-UV region to compare the effect of the missing C-terminal tail on the secondary structure integrity of ALDHTt-508.Both proteins possess negative bands at 208 and 222 cm −1 , typical of a mostly α-helical protein, in accordance with their crystallographic structure as published on PDB (PDB ID: 6FJX and 6FKV for ALDHTtnative and ALDHTt-511, respectively). 30The spectra resemble those published for aldehyde dehydrogenase protein from other sources. 25,31The removal of the C-terminus does not seem to have a major effect on the secondary structure as detectable by Far-UV CD.By quantifying the secondary structure composition of both proteins using computational methods, we identified a 5% loss of β-sheet content from the native protein upon C-terminal tail removal and a proportional increase in disordered structures (Table 5).This minor loss could be a result of the partially denatured or aggregated proteins naturally existing in ALDHTt-508 samples, as detectable by SE-HPLC and DLS. 32In light of these studies,

■ DISCUSSION
The discovery of a thermophilic aldehyde dehydrogenase from T. thermophilus HB27 has led to the elucidation of the role of the C-terminus in ALDH active site control and structural and thermal stability. 15An extension of the C-terminus of other bacterial and archaeal thermophilic proteins has been shown to have a vital role in enzymatic behavior and structural integrity. 6,34,35Determination of molecular features such as these are vital in exploring the mechanisms of thermostability in proteins and of particular interest to the thermophilic enzyme industry.Crystallographic data of the interactions of the ALDHTt-native C-terminus with the overall quaternary structure suggests that it plays a role in thermostability by introduction of strong ionic and hydrogen bonds across the surface of the homotetramer.Ionic bonds and salt bridges have been identified as some of the most influential contributors to protein thermostability, particularly in exposed protein regions, by decreasing the flexibility of surface residues. 36Despite its prominent structure, the extension of the C-terminus has been evolutionarily lost in most ALDH proteins of other thermophilic phylums and of the closely related ALDH from T. thermophilus HB8 (Figure 1).In this research, the successful production of a truncated thermostable ALDH protein, ALDHTt-508 allows for the elucidation of C-terminus involvement in ALDH structural stability and aggregation pathways.The size exclusion HPLC studies show that the intrinsic tetrameric structure of ALDHTt is destabilized in mild conditions, leading to a higher propensity toward formation of precipitates when temperature is increased.These results suggest that the C-terminal extension of ALDHTt holds the tetramer together and acts as an oligomerization aid.Many studies of global protein stability report increased rigidity of the biomolecule as a significant marker of thermostability. 2,37,38s suggested by the modeling of the surface residues in PyMOL (Figure 9), the removal of the C-terminus increases molecule flexibility by exposing hydrophilic residues at the protein core, causing an increase in the dissociation of the tetramer into dimeric and monomeric species (Table 3).
The production of ALDHTt-508 was optimized to account for its lower thermostability.Heat treatment is routinely used as a simple purification procedure to obtain active, homogeneous thermostable enzymes. 39Heat treatment however does not improve yields of the mutant due to effects on the oligomeric state and structural integrity of the protein, as shown in Table 1 and Figure S8.The results from SE-HPLC indicate that loss of the C-terminal extension destabilizes the native tetramer during heat treatment and increases the likelihood of precipitation, most likely by exposure of bondforming residues and increased intermolecular movement.−42 Interestingly, heating of the mutant protein in lysate results in the formation of a hexameric species (∼380 kDa), which is likely to be inactive due to the decrease of enzymatic activity in samples subjugated to 15 min lysate treatment (Figure S8, Table 1).These species are most likely to be a result of trimeric and dimeric species present in the sample, with exposed hydrophobic residues prone to intermolecular linkage, prompting aggregation.Plausibly, prolonged exposure of the recombinant protein in E. coli lysate provided sufficient time for protein unfolding to occur and subsequent proteolysis of susceptible residues by host cell proteases, which have remained active during thermal treatment. 43,44t is clear that removal of the C-terminal extension from ALDHTt affects the protein's thermophilic properties.Optimisation of the purification process revealed a sensitivity of the mutant enzyme to heat-treatment and displayed improved catalytic activity when the heat-treatment step was minimized.Unlike ALDHTt-native, minimization of the heattreatment did not lower yields significantly in production of ALDHTt-508. 15,18Additionally, monitoring the catalytic activity throughout the purification process revealed a minor increase in the function of ALDHTt after the removal of the Cterminal extension, when both proteins are treated to the same purification method.This result is supported by the data displayed in Table 2, which shows improvement of the kinetics of the enzyme toward the substrate hexanal when the Cterminal extension is removed.This validates the theory that the substrate access tunnel of ALDHTt is obstructed by the 22 amino acid long tail. 15The purification profile also suggests that the stability and structure of ALDHTt-508 is retained throughout the purification process, which is performed entirely at room temperature (Table 1).Recombinant ALDHTt-508 therefore holds an economical advantage over production of E. coli aldehyde dehydrogenases which are purified at 4 °C. 45ALDHTt-508 also holds an economical advantage over ALDHTt-native, due to its improved catalytic activity and shorter purification process.Nickle affinity chromatography alongside a 5 min heat treatment step is sufficient for the homogenization of ALDHTt-508, to a purity of 89.9%.Due to the substantial decrease in yield upon gel filtration, the step is deemed as unnecessary unless a high level of protein purity is needed (i.e., for crystallization) (Table 1).
It is worthy to note that although the dissociation into monomers precedes aggregate formation at raised temperatures, it does not seem to impact negatively on the kinetic behavior of the enzyme at working temperatures of 25 and 50 °C (Table 2).In fact, we report a sharp decrease in the K m values for the oxidation of hexanal from 25 to 50 °C, indicating Biochemistry that ALDHTt-508 has a much higher affinity toward the substrate at 50 °C.Surprisingly, the truncated enzyme also shows a much higher affinity toward the substrate than ALDHTt-native, ALDHTt-508, K m = 0.10 ± 0.01 mM, ALDHTt-native, K m = 1.98 ± 0.86 at 50 °C.This is possibly explained by the exposure of the catalytic domain, which is present on each monomer, significantly increasing the amount of hexanal binding to the complex per unit of time. 17Due to the ability of ALDHTt to oxidize aldehydes using both NAD+ and NADP+ as a cofactor, the kinetics of ALDHTt-508 using NADP+ as a cofactor were studied. 17We hypothesized that the opening of the active site would improve the protein's NADP+ mediated activity due to the exposure of NADP+ binding Glu261, in the cofactor binding cleft.Indeed, our results show that there is a minor increase in the specific enzymatic activity of ALDHTt-508 (0.191 ± 0.034 U/mg) when using NADP+ as a cofactor, compared with ALDHTt-native (0.1453 ± 0.0022 U/mg) (Table 2).It is expected that the loss of native tetramer structure reflects in a loss of specific enzyme activity per milligram of active enzyme, as reported for other molecules destabilized by C-terminal truncation; 46 however, this is not the case for ALDHTt-508, as the truncation allows for a more easily accessible active site.This leads to the conclusion that, at the optimum working temperature of the enzyme and in substrate saturated conditions, dissociated dimeric or monomeric species are involved in catalysis or possible reassembly of the native tetramer upon binding of substrate, contributing to its increased enzymatic activity.The formation of precipitates in ALDHTt-508 is temperature-dependent, as evident by the oligomeric stability studies in Table 3, highlighting that the Cterminal tail is paramount in allowing optimum thermostability of the ALDHTt.
The aggregation kinetics of ALDHTt was profoundly altered by the loss of the C-terminus.The destabilization of the molecule at ambient conditions away from the native tetrameric structure induced higher aggregation rates and formation of large insoluble aggregates at 65 and 80 °C (Table 4), as well as accelerating precipitation (Figure 7).This is seen by the higher value of the maximum intensity (I lim ) reached by ALDHTt-508 during heating at both 65°and 80 °C and the lower rate of aggregation (k I ) induced by the presence of the C-terminal extension in ALDHTt-native.This result is easily explainable by the increased mobility of the enzyme upon Cterminal truncation, leading to increased intramolecular interaction. 47The increased mobility is induced by the exposure of positively charged hydrophilic regions present in the substrate access tunnel (Figure 9).The aggregation behavior of human ALDHs and their contribution to disease is well studied; however, there are no studies reported on the aggregation kinetics of thermophilic aldehyde dehydrogenases. 17The homotetrameric ALDH, rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH), follows a similar exponential mechanism of aggregation to ALDHTt, with a critical point of precipitation. 48,49Unlike GAPDH, we found no evidence of tetramer dissociation of ALDHTt-native at elevated temperatures during aggregation kinetic studies, and the aggregates formed can be assumed to constitute unfolded or partially unfolded intact tetramers.Rabbit muscle GAPDH is a mesophilic enzyme with no structural evidence of an oligomer anchoring tail, such as the C-terminus in ALDHTt, which could explain its dissociative aggregation mechanism 50 ALDHTt-508 dissociation into dimeric and monomeric species was inherent to the protein at ambient temperatures.SE-HPLC data dictate that lower molecular weight species are involved during the aggregation and precipitation of ALDHTt-508 (Table 3).
Despite the destabilization of the tetramer by the truncation, the research within this paper also showcases why the Cterminal extension has been evolutionarily excised from ALDH proteins in other organisms.Truncation of the protein by 22 amino acids caused a full opening of the substrate tunnel by the removal of residue Q510, thus severing the bond between the C-terminal and the substrate anchoring region.This is apparent by the substantial increase in activity after purification of ALDHTt-508 (Table 1) and by the large increase in activity at all pH and temperatures tested, when compared with the literature (Figure 4 18 ).The loss in thermostability indicated by the previously determined T m (ALDHTt-native T m = 84 °C, ALDHTt-511 T m = 80 °C) is not reflected in the temperature profile trialled during this study, as the protein is fully active at highest temperature compatible with the aldehyde substrates (50 °C) (Figures 4A and 5).Opening of the homotetramer complex by removal of the C-terminus also increased the substrate specificity toward aldehydes of ALDHTt.In particular, the truncated enzyme gained the ability to catalyze reactions with large and/or cyclic aldehydes such as furfural, citral, o-tolualdehyde, and cyclohexaneboroaldehyde.This can also be explained by the opening of the substrate tunnel upon C-terminus removal, enhancing accessibility of the catalytic thiol to large molecules.
The blocking of the active site by the native C-terminal tail is apparent at all pH levels, as pH profiles reported on ALDHTt show a lower specific enzyme activity of the protein in pH buffers 2−10. 18In fact, ALDHTt-508 performs significantly better at lower pH values, with a 2-fold increase in activity at pH 6 and 7 and a 3-fold increase in activity at pH 5. Additionally, mutant enzyme ALDHTt-508 is active at low-pH citrate buffers, pH 3 and 4, whereas ALDHTt-native shows no reported activity at pH 2−4. 18The truncation of the Cterminus seems to expand the protein's stability toward acidic pH environments (Figure 4B).This result was unexpected due to the apparent loss of stabilizing salt bridges and H-bonds from the structure after the mutation.Other enzymes originating from the T. thermophilus bacterium are also prone to activity loss at acidic pHs, making the mutant unique in its pH profile. 51,52One explanation for the apparent improvement in catalytic efficiency at low pH could be the exposure of negatively charged surface residues which previously interacted with the C-terminal extension, altering the overall surface charge of the protein. 53,54This is demonstrated by the analysis of the surface charges calculated by using the APBS (Adaptive Poisson−Boltzmann Solver) software available in PyMOL (Figure 9).Although an excess of net charges may decrease the overall conformational stability of protein molecules, it has been shown to decrease aggregation rates by electrostatic repulsion between surface charges. 24he finding implies that the mutant ALDHTt-508 is a better contender for applications in aldehyde and alcohol catalysis reactions which require an acidic pH.Further investigation is needed for the protein's stability over time at acidic pH, as the aggregation kinetics studied within this paper were conducted only at pH 8.0.Under acidic conditions, the effect of electrostatic repulsion may improve the aggregation kinetics reported in this study.

Biochemistry
In conclusion, an ALDH from Thermus thermophilus was successfully produced without its native C-terminal extension to probe the impact of the truncation on substrate affinity, pH and temperature stability, oligomeric stability, and aggregation kinetics.Results obtained in this study testify that the removal of the C-terminus broadens the substrate specificity of the enzyme to include a broader range of cyclic/large aldehydes by opening of the active site.The oxidoreductase activity of ALDHTt is substantially increased at acidic pH values by the exposure of negative surface charges.The results herein show that the kinetics of ALDHTt aggregation do not involve the formation of stable intermediates or dissociated states and therefore follow a monomolecular reaction.The C-terminal extension is also shown to increase molecular rigidity by shielding polar residues involved in catalysis.

Figure 1 .
Figure 1.Sequence alignment of the C-termini of thermophilic and mesophilic aldehyde dehydrogenases.Alignment of sequences was performed using Clustal Omega and displayed using GeneDoc software.Purple areas represent regions of highly conserved homology.The blue dashed box displays the characteristic 30 amino acid tail of ALDHTt.

Figure 2 .
Figure 2. Relationship between the substrate access tunnel and C-terminal tail in ALDHTt-native (A) and the ALDHTt-native tetrameric structure as a ribbon model (B).All four monomers of the structure are labeled in the surface model of ALDHTt-native in (C).The monomers are labeled as follows; A, blue; B, pink; C, green; and D, cyan.(D) Repulsion forces between Lys105 (shown in magenta) and the "hook" of the tail, causing the tail to extend toward the N-terminus of the relative monomer.Panel (A) was adapted from ref 15.Available under a CC-BY 4.0 license.Copyright 2018 Kevin Hayes et al.

Figure 4 .
Figure 4. Temperature (A) and pH (B) profiles of ALDHTt-508 using an NAD+ coupled assay and hexanal as the substrate.

Figure 5 .
Figure 5. Screening of ALDHTt-508 aldehyde specificity at 25 °C (A) and 50 °C (B).The enzyme has a broad substrate specificity toward a range of aldehydes, particularly at higher temperatures.

Figure 6 .
Figure 6.Effect of the loss of the C-terminal extension on the aggregation profiles of ALDHTt-native and ALDHTt-508 measured by dynamic light scattering (DLS).The intensity and hydrodynamic diameter data was collected using the Zetasizer Nano-ZSP DLS and normalized using Prism GraphPad (Ver 10.0.1) and fit to a plateau, followed by a one-phase association equation.The mean of three independent experiments is presented.

Figure 7 .
Figure 7. Aggregation kinetics of ALDHTt-native (green) and ALDHTt-508 (red) at temperatures of 65 (A) and 80 °C (B), as measured by dynamic light scattering (DLS).The intensity of diffraction is presented as a function of time.The samples were prepared at 800 μg/mL in 10 mM KPO 4 buffer and filter sterilized.The experimental data points (solid lines) show the mean of at least two independent experiments.The result of fitting (eq 3) is shown by dotted lines.I lim is defined as the final point of aggregation that can be detected by DLS.

Figure 8 .
Figure 8. Dependence of hydrodynamic diameter on the intensity of scattered light of 508 ALDHTt-native (A) and ALDHTt-508 (B).The vertical dotted line corresponds to the value 509 of D H at room temperature.

Figure 9 .
Figure 9. Electrostatics of ALDHTt (dimer) and its truncated mutant, ALDHTt-508.The figure shows the dual polarity of the C-terminal tail (black dashed circle) and the surface charges exposed upon its removal.All ligands from ALDHTt-native and ALDHTt-508 crystallographic data have been removed for clarity (PDB: 6FJX and 6FKV, respectively).Figures were modeled using the Adaptive Poisson−Boltzmann Solver (APBS) available on PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrodinger, LLC).29

Table 2 .
Calculated Kinetic Parameters for the Mutant Enzyme ALDHTt-508 and the Native Enzyme ALDHTt-native Using Hexanal as the Substrate and NAD+ or NADP+ as the Cofactor a

Table 3 .
Distribution of Oligomeric States and Steady-State Hydrodynamic Diameter (D H ) of Native and Truncated Aldehyde Dehydrogenase (ALDH) d aThe hydrodynamic diameter (D H ) was determined by DLS.b Larger soluble aggregates were visualized in only one experiment.c The hydrodynamic diameter corresponds only to the tetrameric population of ALDHTt-508.d Oligomeric states were detected by SE-HPLC in triplicate.The control refers to untreated protein, injected directly after thawing.