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. 2020 Oct 20;5(43):28001–28011. doi: 10.1021/acsomega.0c03521

Conserved Calcium-Binding Residues at the Ca-I Site Involved in Fructooligosaccharide Synthesis by Lactobacillus reuteri 121 Inulosucrase

Thanapon Charoenwongpaiboon †, Panachai Punnatin ‡, Methus Klaewkla ‡,§, Pratchaya Pramoj Na Ayutthaya ∥, Karan Wangpaiboon §, Surasak Chunsrivirot ‡,§, Robert A Field ⊥, Rath Pichyangkura §,*
PMCID: PMC7643167  PMID: 33163783

Abstract

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Inulosucrase is an enzyme that synthesizes inulin-type β-2,1-linked fructooligosaccharides (IFOS) from sucrose. Previous studies have shown that calcium is important for the activity and stability of Lactobacillus reuteri 121 inulosucrase (LrInu). Here, mutational analyses of four conserved calcium-binding site I (Ca-I) residues of LrInu, Asp418, Gln449, Asn488, and Asp520 were performed. Alanine substitution for these residues not only reduced the stability and activity of LrInu, but also modulated the pattern of the IFOS produced. Circular dichroism spectroscopy and molecular dynamics simulation indicated that these mutations had limited impact on the overall conformation of the enzyme. One of Ca-I residues most critical for controlling LrInu-mediated polymerization of IFOS, Asp418, was also subjected to mutagenesis, generating D418E, D418H, D418L, D418N, D418S, and D418W. The activity of these mutants demonstrated that the IFOS chain length could be controlled by a single mutation at the Ca-I site.

Introduction

Inulosucrase (E.C. 2.4.1.9) is a fructosyltransferase, which is found in bacterial species such as Leuconostoc citreum,1Lactobacillus reuteri,2Lactobacillus johnsonii,3Lactobacillus gasseri,4−6 and Streptomyces viridochromogenes.7 This enzyme converts sucrose to inulin-type fructooligosaccharide (IFOS) and β-2,1 fructan, a well-known prebiotic used in the food and pharmaceutical industries. Production of IFOS by inulosucrase has been reported using both free and immobilized enzymes.3,8 Because the prebiotic and immunomodulatory effects of IFOS are largely dependent on its molecular weight,9,10 rational mutagenesis of inulosucrase has been performed to control the product chain length.11−13 Interestingly, the substitution of amino acid residues located in the substrate-entry channel of L. reuteri 121 inulosucrase (LrInu) (Asp479, Ser482, Arg483, Asn543, Trp551, Asn555, Asn561, and Asp689) substantially affects the size distribution of the IFOS produced. The degree of polymerization (DP) of IFOS synthesized by variant enzymes correlated with the distance of the mutated residues from the LrInu catalytic site, suggesting their involvement in oligosaccharide-substrate binding.11

Previous studies demonstrated that LrInu crucially required Ca2+ for its stability and activity,3,14—a feature found in some other carbohydrate-active enzymes.15−17 Based on the crystal structure of L. johnsonii NCC533 inulosucrase (InuJ), two metal ion-binding regions were identified.14 Although the electron density around the metal ion is unclear, site-directed mutagenesis and enzyme activity assays, in both the presence and absence Ca2+, indicate that these residues most likely coordinate Ca2+ and thus define the calcium-binding sites.14 The first calcium-binding site (Ca-I) is located near the enzyme active site, and the conserved residues around Ca-I form a hydrogen-bonding network to some residues in the substrate-binding subsite +1. These residues in Ca-I are conserved in the other homologue enzyme, such as levansucrase.14 This suggests that Ca-I may play a role in oligosaccharide substrate binding and subsite stabilization and indirectly influence catalysis.18 In contrast, the second site (Ca-II) is located on the variable C-terminal region of the protein remote from the active site (∼25 c5).18 Site-directed mutagenesis at Asp520, one of the calcium-binding residues in Ca-I of LrInu, resulted in a decrease of enzyme stability and activity.14 Nevertheless, the contribution of other residues in Ca-I, including Asp418, Gln449, and Asn488, to LrInu stability and activity has not been explored. Some residues at the Ca-I site are positioned close to the predicted oligosaccharide-binding site of the LrInu, and they may play a role in substrate binding and/or IFOS product size distribution.

Herein, we investigated the role of the conserved calcium-binding residues on the stability, activity, and product pattern of the LrInu by using site-directed mutagenesis. The secondary structure of wild-type and variant enzymes was analyzed by circular dichroism (CD) spectroscopy. In addition, molecular dynamics (MD) simulation was employed to compare the conformational dynamics of mutants versus the wild type. These data provide important information on the functional role of the calcium-binding site in LrInu, which will be valuable for engineering FOS production and modification.

Material and Methods

Gene Construction, Expression, and Purification of Inulosucrase Mutants

An InuΔ699His construct, expressing a truncated C-terminal L. reuteri 121 inulosucrase (GenBank accession number AF459437),11 was synthesized by Genscript (USA) and used as a parental gene (wild-type) in this study. Site-directed mutagenesis was achieved by the PCR overlapping extension method,19 using oligonucleotide primers as described in Table S1. PrimeStar DNA polymerase (Takara Bio, Japan) was used for DNA amplification. Mutant genes were ligated into pET-21b (Novagen, USA) via Xhol and NdeI restriction sites. Sequence-verified plasmids were then transformed into Escherichia coli BL21 (DE3) (Invitrogen, USA) for protein expression. The transformant was cultured in an LB broth (supplemented with 100 μg/mL ampicillin, 0.5% (w/v) glucose and 10 mM CaCl2) in an incubator shaker at 200 rpm at 37 °C. Bacteria were cultured to an OD600 nm of 0.4–0.6 before IPTG was added to a final concentration of 0.1 mM. After overnight incubation at 37 °C, bacteria were harvested by centrifugation at 5000g for 10 min. The supernatant was discarded, and the pellets were resuspended in buffer A (25 mM phosphate buffer, 20 mM imidazole, 500 mM NaCl, pH 7.4) and lysed by ultrasonication. Then, the cell debris was removed from crude enzymes by centrifugation at 15,000g for 40 min. The resulting enzyme extracts were stored at 4 °C for further purification.

The recombinant enzymes were purified with a HisTrap HP nickel column (5 mL, GE Healthcare, USA) that was pre-equilibrated with buffer A. The crude enzyme was loaded onto the column and then washed by using buffer A. The purified enzyme was eluted with buffer B (25 mM phosphate buffer, 500 mM imidazole, 500 mM NaCl, pH 7.4). The quality of the purified protein was checked by SDS-PAGE (Figure S1), and the quantity of its concentration was determined by Bradford assay.

Enzyme Activity Assay

The enzyme activities of wild-type and mutant LrInu were determined by using 500 μL of 250 mM sucrose in 50 mM acetate buffer, adjusted to pH 5.5, and 1 mM CaCl2. The reaction was conducted at 50 °C for 10 min and terminated by adding 15 μL of 1 M NaOH. The concentrations of glucose and fructose released from sucrose were determined by a d-fructose/d-glucose assay kit (Megazyme, Ireland). One unit of the enzyme was defined as the amount of enzyme required to release one mmol of glucose and fructose per minute. Transglycosylation activity was determined by the difference between total activity and hydrolysis activity.

Enzyme Kinetics

The wild-type and mutant enzymes (4 μg/mL) were incubated in 500 μL of the substrate solution, containing 5–500 mM sucrose, 50 mM acetate buffer pH 5.5, and 1 mM CaCl2 at 50 °C. The reactions were terminated by adding 15 μL of 1 M NaOH, and the amounts of released glucose and fructose were determined as described above. To determine kinetic parameters, the specific activity of each enzyme versus sucrose concentration was plotted and fitted to either the Hill or Michaelis–Menten equations via OriginPro 2017 software.

CD Spectroscopy

The secondary structure of wild-type and mutant inulosucrase was determined by CD spectroscopy using a CHIRASCAN CD spectrophotometer (Applied Photophysics, UK). Proteins were concentrated to 0.1 mg/mL in 10 mM phosphate buffer (pH 6.0). The spectra were analyzed in the wavelength range 180–260 nm at 30 °C using a 1 mm cuvette. The protein secondary structure was determined using Dichroweb.20,21

Thermostability Determination by Dynamic Light Scattering

The thermostability of wild-type and the mutant enzymes was determined by dynamic light scattering (DLS) using a Dynapro Titan (Wyatt Technology, USA). The proteins (0.2 mg/mL) were heated with a gradient temperature of 40–75 °C at 10 °C/min, and the DLS intensity versus temperature curves were fitted with the Boltzmann equation to determine the melting temperature (Tm) of the mutants using the OriginPro 2017 software.

FOS Production and Analysis

FOSs including inulin were synthesized according to the method described previously.11 Inulosucrases (5 U/mL) were incubated with 500 mM sucrose in 50 mM acetate buffer (pH 5.5) and 1 mM CaCl2. The reactions were performed at 30 °C and 50 °C for 24 h and then terminated by boiling for 10 min. The resulting reaction mixtures were stored at −20 °C until analysis.

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was operated by an ICS 5000 system (Thermo Scientific, USA) fitted with a CarboPac PA 100 column (2 × 250 mm, Thermo Scientific, USA). FOS products were detected and separated by using a linear gradient of 0–500 mM sodium acetate in 150 mM NaOH at a flow rate of 0.25 mL/min for 35 min.

MALDI-TOF: the degree of polymerization of IFOS products was evaluated by MALDI-TOF mass spectrometry (Autoflex speed, BRUKER, USA) using 2,5-dihydroxybenzoic acid (DHB) as the matrix.

Multiple Inulosucrase Protein Sequence Alignment and Conserved Residue Predictions

TheL. reuteri 121 inulosucrase (LrInu) sequence [GenBank ID: AAN05575.1] was selected for multiple sequence analysis and conservation score calculation via the ConSurf server.22 A homolog searching approach was employed with a cut-off E-value of 0.0001 to determine whether homologous sequences were closely related to the LrInu sequence using the HMMER algorithm. Based on 95% maximal and 35% minimal sequence identity to filter out redundant sequences, including uncharacterized inuloscurase sequences, only 58 homologous sequences were chosen for further analyses (Supporting Information). All selected sequences were then computationally aligned and employed to estimate conservation scores from their amino acids by using MAFFT-L-INS-i and Bayesian methods, respectively. Finally, all potential structurally and functionally conserved residues were defined based on catalytic efficiency and conserved structure, respectively, and other residues were predicted as generally necessary residues from their conservation score. Finally, functionally and structurally conserved residues were selected to investigate their impacts on LrInu stability and activity.

Inulosucrase Structure Preparation and MD Simulations

A homology model of L. reuteri 121 inulosucrase was constructed from residues T176 to K698 by the SWISS-MODEL server. The crystal structure of L. johnsonii NCC533 inulosucrase (InuJ; PDB ID:2YFS, 74.2% sequence identity) was used as a template. To assess the quality of the homology model, the RAMPAGE server was employed to generate Ramachandran plots of the model. Most residues were found in favored regions (93.5%) or allowed regions (5.8%) of the plot, indicating the good quality of the homology model. The structure was then protonated at the experimental pH of 5.5 by using the H++ server.23 N- and C- terminal ends were capped using the acetyl beginning group and N-methylamine ending group, respectively. Subsequently, the LEaP module in AMBER18 was employed to add all missing hydrogen and other atoms based on AMBER ff14SB force field parameters. To create amino acid mutations, D418A, Q449A, N488A, and D520A mutants were generated by using the LEaP module.

Each of the structures, including the wild-type system, was immersed in an isomeric truncated octahedral box of transferable intermolecular potential 3 point (TIP3P) water molecules with a buffer distance of 13 c5. Sodium ions (Na+) were added to neutralize the systems. AMBER18 was employed for structural minimizations and MD simulations. A five-step minimization procedure was employed, and all steps included 1000 conjugate gradient steps. First, heavy atoms of the protein were restrained with a force constant of 10 kcal/(mol c52). Then, force constants of 10, 5, and 1 kcal/(mol c52) were applied to restrain all backbone atoms of the protein. Finally, the entire system was minimized without any restraints. MD simulations were carried out under a periodic boundary condition using the PMEMD module of AMBER18. All bonds covalently linked to hydrogen atoms were constrained by the SHAKE algorithm in each simulation, allowing the time step of 0.002 ps. To simulate nonbonded and long-range electrostatic interactions, the particle mesh Ewald method was applied with a cut-off distance of 12 c5. To control the temperature during simulations, Langevin dynamics with a collision frequency of 1.0 ps–1 was applied. Each simulation system was heated from 0 to 303 K for 200 ps in the NVT ensemble, and all backbone atoms were restrained with a force constant of 10 kcal/(mol c52). Next, the systems were equilibrated in the NVT ensemble for 300 ps without restraints. Finally, the systems were simulated for 100 ns in the NPT ensemble at 303 K and 1 atm. During the NPT ensemble, the pressure was controlled by an isotropic position scaling algorithm with a relaxation time of 2 ps.

To determine each system stability, root-mean-square deviations (rmsds) of the backbone atoms and Ca2+ binding residues including Ca2+ atoms were calculated. Because the rmsd values of all systems were stable around 80–100 ns (Figure S2), these trajectories were used for further analyses. Finally, important residues that were previously reported as Ca2+ binding residues and the subsites −1, +1, and +2 residues were used for hydrogen bond analysis of the wild-type and mutant enzymes.

Results and Discussion

The residues in the calcium-binding site I (Ca-I) of LrInu were predicted to be functional residues.

The 3D structure of LrInu comprises two calcium-binding sites, the first of which (Ca-I) is positioned near the active site of the enzyme, at the equivalent position of bound calcium ions in levansucrase,14 suggesting that this site is important for fructansucrases. A second calcium-binding site (Ca-II) is located behind substrate-entry pocket in LrInu, relatively far from the active site;18 this second site has not been found in levansucrases. Amino acid sequence analysis by the ConSurf server revealed that the residues in Ca-I, Asp418, Gln449, Asn488, and Asp520 were highly conserved among the fructosyltransferase (≥90% conservation score) and were also predicted to be functional residues. At the same time, Asn317, Asp659, Ile661, and Ser666, located at Ca-II, were not conserved (Figure 1). The ConSurf server predicts the amino acid signature that is important for the conserved structure or function. Using this approach, our previous study showed that mutation at the predicted oligosaccharide-binding residues, which were also predicted to be functionally conserved residues (Figure 2A–B), strongly affects the IFOS chain length distribution.11 Therefore, in this study, mutational analyses of functionally conserved Ca-I residues (Figure 2C) were performed to investigate their effect on the stability, activity, or product spectrum of LrInu.

Figure 1.

Figure 1

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Representation of predicted conserved residues important for L. reuteri 121 inulosucrase functionality and stability. Prediction of conserved functional (pink), structural (pale blue), and catalytic (red) amino acids including binding site (−1, +1, and +2) and calcium-binding site (I and II) residues, and other residues (x-axis), suggesting significant impacts on LrInu by %conservation score (y-axis).

Figure 2.

Figure 2

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(A) LrInu surface representation of predicted functionally and structurally conserved residues. (B) LrInu surface representation of residues including calcium-binding residues and other important residues for product specificity as previously reported. (C) Close-up view of two superimposed Lactobacillus inulosucrase catalytic sites; LrInu (from 3D modelling, white) and InuJ (2YFT, pale blue). The Ca-I residues were shown in green, the catalytic triad were shown in red, and some acceptor binding residues were shown in purple.

Mutation at the Ca-I Affects Stability, Activity, and Product Profiles of LrInu

Alanine substitution for key residues in LrInu Ca-I, coupled with enzyme activity assessment, demonstrated that Ca-I mutations affected both hydrolysis and transglycosylation activities of LrInu (Figure 3A, Table S2). Substitution at Asp520 and Asp418 by alanine substantially decreased the catalytic activity of LrInu, with these mutants exhibiting only 25 and 38% of wild-type activity, respectively. These residues are physically located close to Asn419 and Glu521, which were predicted to be part of the oligosaccharide-binding sites of LrInu.11 In contrast, Q449A and N488A have a relatively minor effect on the overall catalytic activity of the LrInu. Based on the known structure of inulosucrase, the calcium-binding residues are not involved in the substrate entry and catalytic sites. Therefore, this study provides understanding about the overall catalytic activity of LrInu and the impact of the calcium-binding site.

Figure 3.

Figure 3

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Effect of mutation at Ca-I on biochemical properties of inulosucrase. (A) Hydrolysis (H) and transglycosylation (T) activity of mutant inulosucrase. (B) Thermostability of mutant inulosucrase analyzed by DLS. (C,D) HPAEC analysis of oligosaccharide products synthesized by wild-type and mutant LrInu at (C) 30 and (D) 50 °C.

The effect of Ca-I mutation on LrInu stability was studied using DLS. This technique has been used for determination of protein thermostability by measuring the scattering intensity as a function of temperature24−26 (Figure 3B). When the temperature was elevated, the DLS intensity of mutant LrInus increased significantly, indicating that the proteins denature/aggregate. To determine the melting temperature (Tm) of mutant enzymes, the DLS intensity versus temperature curves were fitted with the Boltzmann equation. The results showed that Tm of LrInu deceased after mutagenesis (Table S3), indicating that the mutant enzyme has lower stability, in line with a previous study.14 The Tm values of D418A (62.6 °C) and D520A (63.8 °C) were slightly lower than that of the wild type (64.2 °C), whereas N488A (61.1 °C) was significantly less stable. However, Q449A has a comparable Tm to wild type (64.2 °C). Overall, these findings are in agreement with previous observations on L. reuteri levansucrase, which show that the mutation at the calcium-binding residue affects the stability and activity of enzyme.14

Calcium ions promote the activity of many bacterial fructosyltransferases, including Bacillus subtilis levansucrase,27Bacillus licheniformis levansucrase,28L. reuteri levansucrase,14 and Streptococcus salivarius fructosyltransferase.29 Ozimek et al.14 reported that mutation of fructosyltransferase calcium-binding residues, both in levansucrase and inulosucrase, resulted in a reduction of the affinity for Ca2+, which may affect the folding of the enzyme.27 Furthermore, based on the crystal structure of inulosucrase, many conserved residues around the Ca2+ binding site form a hydrogen-bonding network to the residues near substrate-binding subsite +1.18 Also, this hydrogen-bonding network links to the general acid/base residue at the active site. Hence, it is possible that mutation of residues in the Ca-I site may change amino acid sidechain conformations and thus affect catalytic activity and stability of the inulosucrase.

The contribution of Ca-I to product distribution patterns for LrInu and mutants thereof at 30 and 50 °C was explored by HPAEC-PAD. These studies provide important observations, showing that mutations at the LrInu Ca-I site affect the IFOS product degree of polymerization (size), while still preserving the linkage regio- and stereo-chemistry of the FOS products (Figure 3C–D). MALDI-TOF MS spectra showed that all LrInu mutants produced a shorter range of IFOS compared to wild type (Figure S3). At 30 °C, D418A produced the shortest range of IFOS (up to DP 12), while D520A produced moderate chain length (DP 16) material. Meanwhile, Q449A and N488A produced the most extended length IFOS among mutant enzymes (up to DP 21). However, for reactions at 50 °C, the trend is slightly different: N488A produced shorter chain length IFOS than Q449A, suggesting that N488 is more sensitive to temperature than Q449. As mentioned above, mutation at Ca-I may affect the conformation and hydrogen-bonding network involving residues in the oligosaccharide-binding site. This might disturb the binding of substrate/acceptor to the enzyme. Not surprisingly, compared to Q449A and N488A, the substitution of Ala at D418 and D520 had much a stronger effect on the product pattern because these residues are positioned adjacent to the oligosaccharide-binding residues, N419 and E521.

Kinetic Parameters for Wild-type and Ca-I Mutants of Lrlnu

Kinetic parameters for wild-type and Ca-I mutant LrInu were determined from plots of enzyme activity versus sucrose concentration,30 fitted using Michaelis–Menten and Hill models (see also Supporting Information, Figure S4). The results show that D418A, Q449A, and N488A exhibit similar kinetic behaviors to the wild-type enzyme, where the total activity (VG) best-fitted the Hill model, while the transglycosylation (VG-F) and hydrolysis activity (VF) were better-fitted with the Michaelis–Menten equation.11 However, D520A showed a slightly different kinetic pattern because the total activity equally well fitted both the Hill and Michaelis–Menten equations, suggesting that the D520A substitution may affect the cooperativity of LrInu (Table 1).30

Table 1. Apparent Kinetic Constants of Wild-type and Ca-I Mutant LrInua.

kinetic parameters WT D418A Q449A N488A D520A
Michaelis–Menten kcatG s–1 1200 ± 60 520 ± 20 850 ± 30 810 ± 40 380 ± 30
  KmG mM 54 ± 9 100 ± 10 56 ± 7 61 ± 11 178 ± 27
  <keep-together>kcatG/Km</keep-together> mM–1 s–1 22 5.1 15 13 2.1
  kcatF s–1 680 ± 30 360 ± 10 450 ± 20 410 ± 50 250 ± 20
  KmF mM 23 ± 5 78 ± 8 21 ± 4 23 ± 8 170 ± 30
  <keep-together>kcatF/Km</keep-together> mM–1 s–1 29 4.6 21 18 1.5
  kcatG-F s–1 790 ± 160 220 ± 58 740 ± 210 810 ± 260 130 ± 30
  KmG-F mM 350 ± 130 330 ± 160 440 ± 210 470 ± 250 200 ± 110
  <keep-together>kcatG-F/Km</keep-together> mM–1 s–1 2.2 0.66 1.7 1.7 0.64
Hill kcatG s–1 1900 ± 600 1000 ± 220 1200 ± 220 1300 ± 450 430 ± 90
  K50G mM nd 700 ± 480 180 ± 110 nd 240 ± 120
  <keep-together>kcatG/K50</keep-together> mM–1 s–1 nd 1.4 6.9 nd 1.8
  hill factorG   0.57 ± 0.11 0.59 ± 0.05 0.62 ± 0.08 0.58 ± 0.12 0.89 ± 0.13
  kcatF s–1 760 ± 100 410 ± 37 430 ± 30 370 ± 30 240 ± 30
  K50F mM 31 ± 14 120 ± 30 20 ± 4 19 ± 5 150 ± 46
  <keep-together>kcatF/K50</keep-together> mM–1 s–1 25 3.6 22 20 1.6
  hill factorF   0.77 ± 0.20 0.81 ± 0.08 1.1 ± 0.2 1.1 ± 0.3 1.1 ± 0.2
  kcatG-F s–1 nd nd nd nd nd
  K50G-F mM nd nd nd nd nd
  <keep-together>kcatG-F/K50</keep-together> mM–1 s–1 nd nd nd nd nd
  hill factorG-F   nd nd nd nd nd
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a

nd = These kinetic parameters could not be determined because the enzymes were not saturated with sucrose, causing high standard errors with curve fits.

Effect of Sucrose Concentration on Product Elongation by Wild-type and Ca-I Mutant LrInu

The chain length of IFOS synthesized by inulosucrase is dependent on sucrose concentration.31 In this study, the relationship between sucrose concentration and the DP of IFOS was explored. In the case of wild-type LrInu, the DP of IFOS increased gradually, from DP 3 to DP 10, when the sucrose concentration increased from 10 to 200 mM. However, after the sucrose concentration reached 250 mM, the DP of IFOS jumped to DP 25 and activity seemed to saturate after that (Figure 4A,F). This result suggested that inulosucrase has two modes of reaction, depending on sucrose concentration. At low sucrose concentration, the inulosucrase prefers to act in hydrolytic mode, where the main product of the reaction is a monosaccharide. In contrast, the enzyme shifts to transglycosylation mode when the sucrose reaches “activation concentration”. This indicates that the modes of action of LrInu are switchable between hydrolysis and transglycosylation. Similarly, for both Q449A and N488A, the DP of IFOS product slowly increased at low sucrose concentration and sharply increased at high sucrose concentration (Figure 4C,D,F). In contrast, the DP of IFOS synthesized by D418A and D520A increased slowly and did not saturate, even when the sucrose concentration reached 750 mM (Figure 4B,E,F). This indicates that the switchability of the LrInu action modes may have been disturbed by these mutations. Because these residues are located near oligosaccharide-binding subsite +2, mutation may affect the oligosaccharide binding affinity. In comparison, mutation at other residues around the catalytic cavity, such as N543A and R483A, did not affect the switchability of enzyme activity mode, although they synthesized a very short range of IFOS (Figure 4F). This result suggests a key interplay between the Ca-I site and IFOS product elongation, which has not previously been reported for inulosucrases.

Figure 4.

Figure 4

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Effect of sucrose concentration on degree of polymerization (DP) of IFOS synthesized by wild-type abd Ca-I mutant LrInu. (A-E) HPAEC chromatograms of IFOS synthesized from various sucrose concentrations (10–750 mM) using (A) wild-type, (B) D418A, (C) Q449A, (D) N488A, and (E) D520A LrInu. (F) Relationship between sucrose concentration and maximum DP of IFOS synthesized by wild-type and mutant LrInu.

Circular Dichroism and Molecular Dynamic Simulation

Based on the 3D structure of the homology model of LrInu, Ca-I is located near the oligosaccharide-binding pocket, which could affect the interaction of a substrate/product to the enzyme indirectly. Substitution at Ca-I residues may indirectly affect the stability, activity, and the product pattern of LrInu via changes in protein conformation or amino acid sidechain orientation. To address this hypothesis, the secondary structure of wild-type and mutant LrInu was analyzed using CD spectroscopy (Figure 5). The results show that the patterns of CD spectra of wild-type and Ca-I mutants LrInu are very similar, indicating that the mutations did not affect the overall secondary structures of enzymes.

Figure 5.

Figure 5

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CD spectra and the predicted contents of secondary structures of wild-type and mutant LrInu.

To complement the CD studies, MD simulations of wild-type and mutant LrInu were performed to elucidate the local conformational changes that impact H-bond networks and the orientation of amino acid residues located near Ca-I. Although the electron density around Ca2+ is unclear, it is reasonable to perform in silico analysis of inulosucrase in the presence of Ca2+ because evidence shows that inulosucrase requires Ca2+ for its stability and activity.14,30 Also, mutation at one of metal binding residues (D520N) dramatically reduced the affinity of Ca2+ bound to the enzyme.14 MD simulation was performed for 100 ns, and the structures that were closest to the average structures of the 80–100 ns MD trajectories were used for analysis. The structures from MD were superimposed with the crystal structure of L. johnsonii NCC533 containing 1-kestose (2YFT) in order to compare the position of amino acids around the active site (using α-carbons of amino acids as a reference atom). In the case of wild-type LrInu, the average position of amino acid residues around the active site was comparable with those of the crystal structure. Likewise, the positions of amino acid residues in the active site of Q449A and N488A mutants are very similar to the available crystal structure. In contrast, alanine substitution at Asp418 and Asp520 caused changes in the position of residues around the oligosaccharide-binding track, such as Asn419 (Figure 6A-E). These results may explain why D418A and D520A mutants possess really low activity and synthesize short-chain IFOS compared with Q449A and N488A mutants.

Figure 6.

Figure 6

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Superimpositions of the InuJ crystal structure (white) and LrInu structure from MD [derived from the structures that are closest to the average structures of the 80–100 ns MD trajectories; (A) wild type; (B) D418A; (C) Q449A; (D) N488A; and (E) D520A; shown in blue]. The green and white ball is Ca2+ bound on LrInu and InuJ, respectively. The red arrows indicated the change in position of amino acid α-carbon between InuJ crystal structures and LrInu structures from MD.

In addition, mutations at Ca-I affected both the number and percent occupancy of H-bonds linking between Ca-I residues and the oligosaccharide-binding site. The previous MD study suggested that H-bond networks indirectly facilitated the binding of the acceptor oligosaccharide of levansucrase.32

According to Pijning et al.,18 Ca2+ may be involved in the flexibility of residues near substrate-binding subsite +1 of inulosucrase from L. johnsonii NCC533 (InuJ). Mutation of Ca-I residues probably affects Ca2+ binding and thus affects the stability of this region of the protein. Because some oligosaccharide-binding residues are located on the same 414–425 loop as Ca-I residues, the instability of this loop may affect oligosaccharide substrate binding, overall enzyme activity, and product size distribution.

Ca-I May be Useful for LrInu Engineering to Achieve Control of IFOS Chain Length

As mentioned above, Ca-I may play an important role in stabilization of the acceptor oligosaccharide-binding site in the LrInu. Substitution of Ca-I residues by Ala, which cannot interact with Ca2+, strongly reduced the DP of IFOS product, possibly because of the conformational instability of the 414–425 loop. For this reason, it is possible that the substitution of these residues by other amino acids, which may weakly bind to Ca2+, may provide different results. Therefore, D418 of the LrInu was replaced by Glu, His, Leu, Asn, Ser, and Trp in order to explore the role of D418-calcium ion interactions in the biochemical characteristics of LrInu. As a result, substitution by Glu and Asn significantly reduced the total activity (Figure 7A) and turnover (Table S4) of LrInu, while the hydrolysis/transglycosylation slightly increased compared with wild type (Table S2). Likewise, replacement by some basic (His), polar (Ser), and hydrophobic (Leu and Trp) amino acid residues strongly decreased the LrInu total activity and increased the hydrolysis/transglycosylation ratio (Table S2).

Figure 7.

Figure 7

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Effect of mutation at D418 on biochemical properties of inulosucrase. (A) Hydrolysis (H) and transglycosylation (T) activity of mutant inulosucrase. (B) Thermostability of mutant inulosucrase analyzed by DLS. (C,D) HPAEC analysis of oligosaccharide products synthesized by wild-type and mutant inulosucrase at (C) 30 °C and (D) 50 °C.

For thermal stability studies, the melting temperature of D418 mutants, except D418N, decreased around 1.5 °C (Figure 7B, Table S3). This was attributed to the fact that the D418 residue is essential for both the stability and the activity of the LrInu. The product pattern of D418 mutants was also evaluated by HPAEC and MALDI-TOF MS. At both 30 and 50 °C incubation temperatures, D418N synthesized the most extended chain range of IFOS among all of the D418 mutants assessed in this study (up to DP 18 at 50 °C, and DP 21 at 30 °C), but it was shorter than wild type (DP 23; Figures 7C, S3). On the other hand, D418E, D418H, and D418S produced moderate chain length IFOS (DP 16–17 at 30 °C), while D418A, D418L, and D418W synthesized the shortest range of IFOS (DP 12–15 at 30 °C). This finding correlates with our protein sequence alignment data, which indicated that position 418 of LrInu—an Asp residue—is very highly conserved (conservation score of 88.8%). In comparison, Asn is found much less frequently (11.1%) (data not shown). As mentioned, D418 is located near N419, which forms an H-bond network to other residues around the oligosaccharide-binding pocket. Mutation at D418 should disrupt this H-bond network and may affect the binding affinity to the acceptor/substrate.

Conclusions

Mutagenesis analysis of four conserved calcium-binding resides at the Ca-I site was employed in order to investigate the role of these residues for stability, activity, and IFOS product pattern of LrInu. Although Ca-I is located remote from the critical active site residues required for catalysis, alanine scanning at these residues strongly affects the IFOS chain length produced by LrInu. CD spectroscopy and MD simulations suggest that mutation did not influence the overall structure of the enzyme, but changed the orientation and hydrogen bonding network of oligosaccharide substrate-binding residues. This study provides new insights into the role of calcium-binding sites in stability, activity, and product patterns of LrInu, which should prove useful for inulosucrase engineering.

Acknowledgments

T.C. is thankful to Department of Biochemistry, Faculty of Science, Chulalongkorn University and Department of Biological Chemistry, John Innes Centre. S.C. was partially supported by the Structural and Computational Biology Research Unit, Faculty of Science, Rachadaphiseksomphot Endowment Fund, Chulalongkorn University.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c03521.

  • Homologous sequences used for multiple sequence alignment; list of oligonucleotide primers used for site-directed mutagenesis; SDS PAGE of purified LrInus; RMSD plots derived from MD; activity of wild-type and mutant enzymes; melting temperature of mutant LrInus; MALDI TOF analysis of oligosaccharide synthesized by wild-type and mutant LrInu; kinetic fitting curves of wild-type and mutant LrInus; and apparent kinetic constants of wild-type and Ca-I mutant LrInu (PDF)

Accession Codes

L. reuteri 121 inulosucrase, AF459437 (NCBI).

Author Contributions

T.C., R.P., and R.A.F. conceptualized and planned the study. T.C. performed protein purifications and all enzymic assay and led data analyses. S.C. planned the MD study. P.P. and M.K. performed the MD study. P.P.N.A. planned and performed sequence analyses. T.C., R.P., R.A.F., S.C., P.P., M.K., P.P.N.A., and K.W. wrote the manuscript.

The authors declare no competing financial interest.

Supplementary Material

ao0c03521_si_001.pdf (1MB, pdf)

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Supplementary Materials

ao0c03521_si_001.pdf (1MB, pdf)

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