Enhancing the activity and thermostability of Streptomyces mobaraensis transglutaminase by directed evolution and molecular dynamics simulation

Biochemical Engineering Journal 151 (2019) 107333

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Regular article

Enhancing the activity and thermostability of Streptomyces mobaraensis transglutaminase by directed evolution and molecular dynamics simulation

T

Yihan Liu, Lin Huang, Mengying Shan, Jingcheng Sang, Yanzhen Li, Longgang Jia, Nan Wang, ⁎ ⁎ Shuang Wang, Shulin Shao, Fufeng Liu , Fuping Lu State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, National Engineering Laboratory for Industrial Enzymes, The College of Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Avenue, Tianjin Economic and Technological Development Area, Tianjin, 300457, PR China

H I GH L IG H T S

mobaraensis transglutaminase (MTG) mutant with improved properties was obtained. • ATheS. MTG exhibited a 1.95 times specific activity of wild type at 50 °C. • The MTG mutant showed a 1.66-fold half-life of wild type at 50 °C. • Molecular mutant dynamics simulations clarified the mechanism for improved MTG mutant. • It will be beneficial for rational design to engineer MTG mutants for application. •

A R T I C LE I N FO

A B S T R A C T

Keywords: Streptomyces mobaraensis transglutaminase Directed evolution Specific activity Thermostability Molecular dynamics simulation

Streptomyces mobaraensis transglutaminase (MTG) has been extensively used in food industry and other biotechnological fields due to its cross-linking modification of proteins. Thus a MTG variant with a higher thermostability as well as an enhanced activity is desired because some processing for MTG applications is often performed at higher temperatures. In this study, the activity and thermostability of wild-type MTG (WT) were enhanced via directed evolution using error-prone PCR, and the mutant MTG (E164L) with the improved specific activity and thermostability was obtained using a high-throughput activity assay. E164L exhibited a 1.95 times specific activity of WT at 50 °C. Meanwhile, the half-life of E164L at 50 °C was 1.66-fold of WT. The molecular dynamics (MD) simulation results indicated that the mutation Glu164Leu resulted in the weaker interactions of Asp159-Glu164 and Gly228-Leu231, leading to the enhanced instability of Ile240-Asn253 linked to Gly228Leu231 by eight residues. It further caused the reduced interactions between loop region 1 (Ile240-Asn253) and loop region 2 (His277-Met288), facilitating the access of the substrate molecule to the active site. This study improved the understanding of structure-activity relationship for MTG adapted to high temperature conditions. It also provides theoretical foundation and preliminary information for engineering MTG with enhanced characteristics to meet the industrial requirements.

1. Introduction Transglutaminase (protein-glutamine γ-glutamyltransferase, TG, EC 2.3.2.13) can catalyze an acyl transfer reaction occurred at the γ-carboxyamide groups of peptide-bound glutamine residues (acyl donors) and the ε-amino groups of lysine residues/primary amines (acyl acceptors), leading to the generation of ε-(γ-glutamyl) lysine isopeptide bonds formed intramolecularly or intermolecularly [1–3]. Therefore, it can stimulate the cross-linking and polymerization of proteins [4].

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So far, transglutaminases have been found in many organisms including animals [5,6], plants [7,8], and microorganisms [9–15], and their functions and properties have been characterized. Among them, transglutaminase from Streptomyces mobaraensis (MTG) has been widely exploited as an industrial catalyst due to its broad substrate specificity, independence of calcium ion, and the relatively low molecular weight [2]. The principal application of MTG is to modify the functional properties of proteins in food systems [16]. The reactions initiated by MTG can create profound changes to proteins in food matrices,

Corresponding authors. E-mail addresses: [email protected] (F. Liu), [email protected] (F. Lu).

https://doi.org/10.1016/j.bej.2019.107333 Received 26 February 2019; Received in revised form 24 June 2019; Accepted 6 August 2019 Available online 07 August 2019 1369-703X/ © 2019 Elsevier B.V. All rights reserved.

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2. Materials and methods

resulting in the enhanced texture and stability in association with the temperature, syneresis, emulsifying properties, gelation, and the enhanced water-binding capacity. MTG may even not only render it more nutritious as a result of the possibility of adding essential amino acids but maintain the pH, color, flavor or nutritional quality of food [16]. However, in some applications using MTG to crosslink proteins, they need to be performed at a higher temperature (≥50 °C), resulting in the requirement of a thermostable transglutaminase. Furthermore, MTG with an enhanced specific activity is desired due to the limited activity of MTG by fermentation, which is beneficial to reducing the cost for application and improving the production efficiency [17]. Thus it is of great interest to improve the activity and thermostability of MTG. To this end, rational design is an efficient method to improve certain biochemical and catalytic properties of enzymes by introducing specific and intentional changes to the gene sequences. However, it usually depends on both the availability of the three-dimensional structure of enzyme and information related to the protein sequence, structure, and function [18–21], resulting in the limited studies to improve the properties for enzymes. To overcome these problems, directed evolution is more powerful to enhance the activity and stability. Directed evolution, mainly including error-prone PCR and DNA-shuffling, is generally involved in creating a mutant library, screening and selecting the target mutants with desirable performance without their detailed structural information [22–24]. Up to now, little work has been performed to improve the activity or thermostability of MTG by rational design or directed evolution, or their combination. It was reported that the mutants (S2P, S23 L, Y24 N, G257S, K269E, H289Y, and L294 M) engineered by error-prone PCR indicated a significantly enhanced thermostability at 60 °C, especially S2P increased the half-life (T1/2) by 270% [25]. Then, the seven “hot spots” was modified by saturation mutagenesis combined with DNA-shuffling. The simultaneous mutagenesis of amino acids at positions 23, 24, 269, and 289 led to a MTG variant with almost two-fold higher specific activity and an optimum temperature of 55 °C [26]. Three penta-site MTG variants, S23V-Y24N-K269D-H289Y-K294 L, S2P-S23V-Y24N-K269DK294 L and S2P-S23V-Y24N-S199A-K294 L, were concurrently engineered by site-directed mutation. It was observed that S2P-S23VY24N-S199A-K294 L demonstrated the longest T1/2 at 50 °C and 60 °C [27]. Although the seven “hot spots” have been identified for improving the activity and thermostability of MTG, it is necessary to further explore the effect of key amino acid residues on the characteristics of MTG thoroughly. It would lay the foundation for engineering MTG mutants with the improved activity and thermostability by modification of multiple residues synergy to optimize MTG tertiary structure by rational design. Therefore, MTG was subjected to the strategy of directed evolution to continuously seek new mutants exhibiting the improved activity and thermostability. Nevertheless, the bottleneck for most of directed evolution experiments is short of the efficient high-throughput screening or selection methods for the target property [28,29]. Therefore, it is beneficial to developing generic screening or selection tools to facilitate the process of characterizing novel enzyme properties, and also to bring in much more changes to the function of enzymes [30]. In our previous study, a high-level extracellular expression of MTG was achieved by engineering a novel secretary expression system in Corynebacterium glutamicum ATCC 13032 [17]. Here, MTG variants with higher specific activities and excellent thermostabilities were constructed using error-prone PCR and efficiently selected by the screening method based on the novel C. glutamicum ATCC 13032 expression system without the redundant procedure to lyse Escherichia coli cells expressing MTG. Meanwhile, the enzymatic properties of mutant MTG were investigated. Subsequently, the three-dimensional structure of the mutant MTG was evaluated using molecular dynamics simulation (MD) to explore the molecular mechanism for the high catalytic efficiency and thermostability and to deepen our understanding of the structurefunction relationship on enzymes adapted to the high temperature.

2.1. Plasmids, strains, and media Plasmid pXMJ19L was engineered by inserting the tac-M promoter and signal peptide ΔS0949 into pXMJ19 in our previous study [17]. The tac-M promoter was engineered with sequence TGTGGTACCAT to replace the sequence of the extended -10 region (GCTCGTATAAT) in the tac promoter by overlap extension PCR [31]. It can be induced with isopropyl-β-D-1-thiogalactopyranoside (IPTG) and repressed by the lac repressor [32,33]. The signal sequence ΔS0949 was engineered by replacing +1 leucine with +1 glutamine in signal sequence S0949, which was associated with the twinarginine translocator-type signal sequence [34]. Plasmid pXMJ19L-promtg, containing the coding sequence of pro-domain (45 amino acid residues) and the mature peptide (331 amino acid residues) of MTG gene (promtg) from S. mobaraense, was engineered in C. glutamicum ATCC 13032 by our laboratory [17]. E. coli DH5α and C. glutamicum ATCC 13032 were cultivated in our laboratory for vector construction and protein expression, respectively. E. coli DH5α was grown at 37 °C in Luria-Bertani (LB) medium (Bactotryptone 10 g/L, yeast extract 5 g/L, and NaCl 10 g/L) supplemented with chloroamphenicol (50 μg/mL) when necessary. C. glutamicum ATCC 13032 was cultivated at 30 °C in MMTG medium (60 g of glucose, 1 g of MgSO4, 30 g of (NH4)2SO4, 1.5 g of KH2PO4, 0.01 g of FeSO4·7H2O, 0.01 g of MnSO4·4H2O, 450 μg of thiamine hydrochloride, 450 μg of biotin, 0.15 g of DL-methionine, and 50 g of CaCO3 per liter of distilled water) medium supplemented with chloroamphenicol (10 μg/ mL) when necessary. 2.2. Construction of error-prone PCR library The fragment of promtg gene in pXMJ19L-promtg was used as a template for error-prone PCR. The reaction mixture containing 10 μM forward primer (5′ -CGCGGATCCGGCAGCGGCACCGGGGAAG-3′) and reverse primer (5′-AACTGCAGCGGCCAGCCCTGTGTCACCT-3′) with the restriction sites BamHI and PstI (underlined), 10 ng of template DNA, 5 mM MgCl2, 0.25 mM MnCl2, 0.2 mM dATP, 0.2 mM dGTP, 1 mM dTTP, 1 mM dCTP, and 5 U Taq DNA polymerase, was subjected to PCR under conditions: one cycle: 95 °C, 5 min; 30 cycles: 94 °C, 30 s, 55 °C, 30 s, 72 °C, 1 min; one cycle: 72 °C, 10 min. The purified errorprone PCR product was digested with both BamHI and PstI, and then inserted into the BamHI-PstI-linearized pXMJ19 L with the 6-His tag coding sequence at the 3′end for protein purification. The recombinant plasmids, pXMJ19L-promtgm, were transformed into E. coli DH5α by electroporation. Subsequently, the plasmids extracted from the recombinants were transformed into C. glutamicum ATCC 13032 for protein expression. The resultant clones, harboring the error-prone PCR library, were spreaded on LB agar plates with chloroamphenicol (10 μg/ mL) and incubated at 30 °C for 12 h. 2.3. Screening of the mutant library After overnight cultivation on LB plate, colonies were transferred to 96-well plates containing MMTG medium supplemented with 10 μg/mL chloroamphenicol. The 96-well plates were incubated at 30 °C with shaking at 160 rpm until OD600 reached 0.8, followed by adding IPTG to a final concentration of 0.5 mM. After induction at 20 °C for 16 h, the supernatant of each well was centrifugally collected (6000 rpm, 10 min) and transferred into a new plate. Subsequently, neutral protease (200 μg/mL, Solarbio, China) was mixed with the culture supernatant to remove the pro-domain to activate MTG. After reacting at 37 °C for 1 h, the mixtures were transferred into two identical plates. One plate was directly assayed at 50 °C, whereas the second plate was incubated at 50 °C for 30 min in a moist chamber, and the plate was then cooled on ice for 10 min to measure the activity at 50 °C. Initial activities of WT and mutant MTGs and their residual activities after heat treatment at 2

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50 °C were measured according to the section of “Activity assays of MTG”. The positive mutants with enhanced activities and thermostabilities were selected by comparing the initial activity and the ratio of the initial activity to the residual activity of each mutant, respectively. All experiments were carried out in triplicate.

using AutoDockTools 1.5.6 [43]. Semi-flexible docking allowed the chemical bond of CBZ-Gln-Gly to rotate freely while the coordinates of all atoms in MTG were fixed. The grid parameters were set to build the space (a cubic box of 40 Å × 40 Å × 40 Å) near the three residues (Cys64, Asp255, and His274) of MTG active site [39] as the docking area, and the grid spacing is set to 0.375 Å. Next, the docking parameters were set as following. The total number of conformations obtained by molecular docking was 20, the energy evaluations were the default value of 2500000, the generations, rate of gene mutation and rate of crossover were 27000, 0.02, and 0.8, respectively. Finally, MD simulation was performed by selecting the composite conformation with the lowest protein-substrate docking binding energy in the molecular docking. GROMACS 5.1.4 software was used to perform MD simulation with GROMOS96 54a7 force field parameters [44]. The detailed simulation parameters were the same as described elsewhere [45,46]. With periodic boundary conditions, the minimum distance between MTG and the edge of the cube box was 15 Å, and then the box was filled with water molecules. The SPC/E model was used to describe water. The ions were replaced with the corresponding number of water molecules at random positions to make the system charged zero. The steepest decent method of 50000 steps was used to optimize the simulation systems. Shortrange van der Waals (vdW) interaction and short-range electrostatic interaction (ELE) were smoothly truncated at 10 Å. In the isochoricisothermal (NVT) ensemble, different temperature gradients (40 °C, 50 °C, and 60 °C) were repeated three times. The leapfrog integral algorithm was used to integrate the motion equation with step length of 2 fs for MD simulations. The simulation systems were simulated for 100 ps in isothermal isovolumetric (NVT) ensemble. All covalent bonds involving hydrogen atoms were constrained by LINCS algorithm. Particle Mesh Ewald (PME) method was used to calculate the long-distance electrostatic interactions. The mesh size was 0.16 Å, and its intercept was 12 Å. Protein, CBZ-Gln-Gly, and water molecules were coupled with external temperature and pressure baths. In the isothermal-isobaric (NPT) ensemble, the Parrinello-Rahman barostat and V-rescale thermostat method were used to control the pressure and temperature, respectively. Finally, each simulation was performed for 50 ns, and the coordinates were saved every 2 ps. Several auxiliary programs in GROMACS 5.1.4 package were used to analyze the simulation trajectories. The programs of gmx rmsd and gmx rmsf were used to calculate the root mean square deviation (RMSD) and root mean square fluctuations (RMSF), respectively. The distance and the number of contacts between any two residues were calculated by the gmx mindist program. The secondary structure analysis of MTG was carried out by employing the Dictionary Secondary Structure of Proteins (DSSP) software [47]. The snapshots of the protein structures were made using the Visual Molecular Dynamics (VMD) 9.1.4 software [48].

2.4. Activity assays of MTG MTG activity was measured using the specific substrate, α-NCarbobenzyloxy-Gln-Gly (CBZ-Gln-Gly), by hydroxamate formation described by Liu et al. [17]. One unit of MTG was defined as the amount of enzyme generating 1 μmol l-glutamic acid γ-monohydroxamate per min at 50 °C. 2.5. Expression and purification of MTG WT and mutant E164 L were expressed in C. glutamicum ATCC 13032 harboring the plasmid pXMJ19L-promtg or pXMJ19L-E164L, respectively. The recombinant C. glutamicum ATCC 13032 was inoculated into 2 mL of LB medium supplemented with chloroamphenicol (10 μg/mL) and incubated at 30 °C for 10 h with shaking (200 rpm). One milliliter of start culture was transferred into 50 mL of MMTG medium containing chloroamphenicol (10 μg/mL) with shaking (200 rpm) at 30 °C. When the optical density at 600 nm (OD600) reached 0.8, IPTG was added to a final concentration of 1 mM, and the culture was further induced to express WT and E164 L at 20 °C with shaking at 160 rpm for 40 h. WT and E164 L with the 6-His tag were purified using a nickel-nitrilotriacetic acid (Ni-NTA) agarose gel column according to the method of Liu et al [17]. Protein concentration was determined using the Bradford method with bovine serum albumin (BSA) as the standard [35]. 2.6. Characterization of MTG The optimal temperatures of WT and E164 L were measured by conducting the enzyme assay at different temperatures (20–60 °C) in 10 mM sodium phosphate buffer (pH 6.0) for 10 min. To determine the optimal pH, the activities of WT and E164 L were evaluated in 10 mM sodium phosphate buffer (pH 4.0–9.0) at 50 °C. The activities of WT and E164 L at the optimal temperature and pH were taken as 100%, respectively. All experiments were carried out in triplicate. To determine the thermostabilities of WT and E164 L, they were incubated in 10 mM sodium phosphate buffer (pH 7.0) without substrate at 50 °C for different time (20, 40, 60, 80, 100, and 120 min). The pH stabilities of WT and E164 L were determined by incubating them at 40 °C in 10 mM sodium phosphate buffer with various pH (4.0, 5.0, 6.0, 7.0, 8.0, and 9.0) without substrate for 1 h. After cooling on ice for 10 min, their residual activities were determined using the standard enzyme assay method. The initial activities of WT and E164 L without pretreatment were taken as 100%, respectively. All experiments were carried out in triplicate.

3. Results and discussions 3.1. Screening of mutant library

2.7. Molecular dynamics simulations The screening strategy for mutant MTGs was based on the original activity and residual activity after incubation at high temperature. Approximate 5700 mutants were screened to identify those with enhanced activities and thermostabilities. About 900 mutants showed activities in the first screening for activity, and 23 mutants exhibited enhanced activities. Thereafter 7 mutants from the aforementioned 23 mutants demonstrated better thermostabilities than WT at 50 °C via the second screening for thermostability. The obtained mutants were further cultivated in flasks to characterize the enzymatic properties, 3 mutants with enhanced activities and thermostabilities without compromising their activities were obtained. Compared with the nucleotide sequence of WT, the three mutant MTGs gene indicated two mutation sites, resulting in substitution of GAG→CTG or GAG→TTG in

SWISS-MODEL (https://www.swissmodel.expasy.org/) [36] template library was searched using BLAST [37], and HHBlits [38] for evolutionary related structures matching the target sequence and the coordinates of MTG (PDB ID: 1IU4 [39]) were used as a template for 3D structural model of WT and E164L. The online tools Verify3D (http:// servicesn.mbi.ucla.edu/Verify3d/) [40,41] was used to explore the structural rationality of target protein MTG versus the template protein MTG (1IU4) with a specific crystal structure. The substrate, CBZ-GlnGly, was constructed using Chemoffice software. The topology file of CBZ-Gln-Gly was obtained using the website of Automated Topology Builder version 2.0 (http://atb.uq.edu.au/) [42]. Molecular docking between MTG and CBZ-Gln-Gly was performed 3

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mg). These results indicated that a higher activity is conferred to MTG by the introduced mutation. The improved specific activities of enzymes can enhance their fermentation efficiency and reduce their dosages, which are beneficial to reducing the cost of productions and applications, respectively. Therefore, MTG with the enhanced activity is greatly required for food industry. Here, E164 L exhibited a significantly improved specific activity, indicating that it was a potential MTG candidate for production in a large scale.

nucleotide sequence. It was further revealed by sequencing analysis that the three mutants were located at the same amino acid position (Glu164Leu). Generally, the recombinant enzymes intracellularly expressed in E. coli are generally obtained by sonication to disrupt cells, leading to a time-consuming operation. Therefore, it is essential to engineer an expression system to secrete the recombinant enzymes directly into culture medium, which is beneficial to screening the mutant library. In our previous study, we made use of a secretion signal peptide ΔS0949 and a modified Tac promoter (Tac-M promoter) to construct a plasmid pXMJ19 L in C. glutamicum ATCC 13032 to optimize the secretion system for MTG [17]. Here, this secretion expression system was exploited to simplify the process for screening MTG mutant library.

3.3. Enzymatic property of WT and mutant MTG Both WT and E164L demonstrated the optimum activity at pH 6.0 (Fig. 1b). As shown in Fig. 1c, WT and E164 L exhibited the similar pH performance at pH 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, indicating that there was no significant difference between WT and E164L in the pH stability. The optimum temperature for E164L was found to be around 50 °C, quite similar to that for WT (Fig. 1d), but E164L exhibited ahigher relative activity than WT in the temperature range of 20–60 °C. As shown in Fig. 1e, E164L demonstrated a higher residual activity than WT after treatment at 50 °C. WT retained around 35%, 21%, 11%, and 0% of its initial activity after treatment at 50 °C and pH 7.0 for 60, 80, 100, and 120 min, respectively, but E164L could maintain 60%, 41%, 29%, and 16% of its original activity (Fig. 1e). Meanwhile, the half-life (T1/2) of WT and E164L at 50 °C was also studied. WT showed a T1/2 of

3.2. Expression, purification, and activity assay of WT and mutant MTG After induction at 30 °C for 40 h, significant activities of WT and mutant MTG were detected in the culture supernatant of 13032/ pXMJ19L-promtg and 13032/pXMJ19L-E164L, but no clear activity was found in the supernatant of 13032/pXMJ19 L harboring the empty plasmid pXMJ19 L. After Ni-affinity chromatography, the specific activities of purified WT and E164 L were measured at various temperatures. The specific activity profiles of E164 L was compared with that of WT in Fig. 1a, and the specific activity of E164 L was higher than that of WT from 20 °C to 60 °C. E164 L exhibited the highest specific activity of 52.3 U/mg at 50 °C, approximately 1.95 times of WT activity (26.8 U/

Fig. 1. Specific activities and characterizations of WT (◼) and E164 L (□). (a) Specific activities of WT and E164L at different temperatures. The specific activities for WT and E164L were determined at various temperatures ranging from 20 °C to 60 °C and pH 6.0. (b) Effect of pH on the activities of WT and E164L. The optimum pH for WT or E164L was determined over a pH range of 4.0–9.0 at 50 °C, respectively. The activity of WT or E164L at the optimum pH was taken as 100%, respectively. (c) Effect of pH on the stabilities of WT and E164L. The pH stability was determined by measuring the residual activity of WT and E164L after incubation without substrate at pH 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 and 40 °C for 1 h. The original activity of WT or E164L without preincubation was taken as 100%, respectively. (d) Effect of temperature on the activities of WT and E164L. The optimum temperature for WT or E164L was determined at different temperatures ranging from 20 °C to 60 °C and pH 6.0. The activity of WT or E164L at the optimum temperature was taken as 100%, respectively. (e) Effect of temperature on the and stabilities of WT and E164L. The thermostability was determined by measuring the residual activity of WT or E164 L after treatment without substrate at 50 °C and pH 7.0 for 20, 40, 60, 80, 100, and 120 min. The original activity of WT or E164L without preincubation was taken as 100%, respectively. The data were the average values of three independent experiments and the error bars indicated the standard deviations.

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Fig. 2. Protein sequence alignment of MTG (1IU4) and MTG. The identical amino acids were highlighted in solid black. The different amino acids were highlighted in grey and labelled with blue stars. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

between the two proteins, thus, indicating the high structural similarity. Since there was no 3D conformation of the substrate CBZ-Gln-Gly combined with MTG, a common method was used to obtain a complex of substrate and target protein by molecular docking. Then MD simulations were used to study the structural properties of proteins and their interactions with substrates specifically [54–56]. The substrate CBZGln-Gly was docked with MTG to obtain multiple binding conformations, and the conformations with the top 5 lowest binding energy were selected (Table 1). The lowest value of the binding energy was -1.17 kcal/mol in the conformation 1. Corresponding to the lowest value of the binding energy, the substrate CBZ-Gln-Gly maintained a reasonable distance and angle from three residues (active site) of MTG. The binding of MTG to the substrate was most advantageous, and MTG was most likely to undergo a catalytic reaction in this binding conformation shown in Supplementary Fig. 2. To verify the stability of MTG secondary structure during MD simulation, the changes in distribution of each secondary structure of MTG (1IU4) and MTG were drawn as a function of simulation time, and the average content of each secondary structure was calculated over 50 ns. As exhibited in Supplementary Fig. 3, MTG had a similar secondary structure distribution in comparison to MTG (1IU4). In MD simulation of 50 ns, the secondary structure of MTG was relatively stable, but the secondary structure of a small number of amino acid residues in MTG (1IU4) was not stable (αHelix appeared at the end of MD simulation at the N-terminal simulation and α-Helix disappeared at the beginning of MD simulation at the C- terminus). Considering the high homology between MTG and MTG (1IU4) (93.35%), it indicated that MTG modeled using SWISS-MODEL was not only similar to MTG (1IU4) in the conformation, but also more stable in comparison to MTG (1IU4) in the secondary structure. As demonstrated in Supplementary Fig. 4, the content difference of most secondary structures (Coil, β-Sheet, β-Bridge, and Bend) of MTG and

42.4 min, whereas E164L displayed a T1/2 of 70.5 min, which was about 1.66 times of WT. The aforementioned results demonstrated that the thermostability of E164L was enhanced by the mutation. Recently, MTG has been applied in various potential applications except food processing, such as materials science, textiles and leather processing, tissue engineering, and other biotechnological fields [49–53]. But it would be advantageous to perform the cross-linking reaction of MTG at a higher temperature for some applications. As expected, a mutant MTG with an enhanced thermostability was obtained, which could expand the various industrial applications for MTG. 3.4. Molecular mechanism of improved activity of E164L analyzed by molecular dynamics simulations As shown in Fig. 2, the sequence search results indicated that the homology of MTG to MTG (1IU4) was 93.35%. Therefore, the target sequence could be modeled based on MTG (1IU4) due to the high homology between the target protein and the template protein. To further confirm the rationality of the homology modeling of MTG secondary structure, the averaged 3D-1D score of each amino acid residue of MTG (1IU4) and MTG was calculated. The Ramachandran plots of MTG (1IU4) and MTG were drawn using an additional analysis tool within VMD. As demonstrated in Supplementary Fig. 1, 92.15% amino acid residues in MTG had an averaged 3D-1D score of more than 0.2. While in MTG (1IU4), 91.24% amino acid residues had an averaged 3D1D score of more than 0.2, indicating almost the same value. When the averaged 3D-1D score exceeded 0.2, the structure of the region could be reasonable [40,41]. These data indicated that the homology modeling structure of the target sequence was reasonable. As indicated in Fig. 3, the distribution range and density of the φ/ψ dihedral angle of mainchains of residues in MTG were highly similar to that of MTG (1IU4). It proved that there was a similar mainchain extension orientation 5

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Fig. 3. Ramachandran plots of MTG (1IU4) (a) and MTG (b). Table 1 The binding energies between CBZ-Gln-Gly and MTG. Serial number

Autodock binding energy (kJ/mol)

1 2 3 4 5

−1.17 −0.9 −0.47 −0.42 0

MTG (1IU4) was within 0.5% in MD simulation of 50 ns. Only in the structures of Turn and Helix, the difference in content between MTG and MTG (1IU4) was more than 1%. Considering that there was about 6% homology difference between MTG and MTG (1IU4), it could be concluded that 3D conformation of MTG modeled using SWISS-MODEL and its secondary structure was reliable. To investigate the effect of the mutation in residue 164 on the overall structure of MTG, RMSF values of both WT and E164 L were calculated at 60 °C. Compared with WT, E164 L had an observable increase of RMSF value for the two regions of Ala155-Ala166 and Ile240Asn253 (Fig. 4a). It was clear that Leu164 residue of E164 L was located exactly in the first region (Ala155-Ala166) where RMSF value was larger than that of WT, indicating that the stabilities of two regions (Ala155-Ala166 and Ile240-Asn253) were destabilized by the mutation Glu164Leu. However, the regions Ala155-Ala166 and Ile 240-Asn253 only accounted for a small fraction of MTG. If MTG stability was fully analyzed, the value of RMSD could characterize the overall stability of MTG as a function of simulation time. As indicated in Fig. 4b, although the stability of regions Ala155-Ala166 and Ile240-Asn253 was reduced, the values of RMSD between WT and E164L were not significantly different in the whole MD simulation. It was particularly noteworthy that the value of RMSD of E164L tended to be stable after 10 ns, and its value was about 4 Å at the end of MD simulation. While the RMSD value of WT was increased throughout MD simulation. Its value was approximately 5 Å at the end of the MD simulation, which exceeded that of E164L at the same time. As exhibited in Fig. 1e, the thermostability difference of WT and E164L was obvious when the experiment was carried out for more than 10 min. Namely the thermostability of E164 L was higher than that of WT, and the duration of MD simulation was generally in the nanosecond range. Therefore, the results of MD simulation were not completely compatible with the experimental data. However, from the trend indicated in Fig. 4b, the difference of stability between WT and E164L would become larger with the extension of simulation time and eventually data were similar to that in Fig. 1e. Subsequently, in order to match the situation at the optimum temperature for WT and E164 L, the trajectory at 50 °C was used as the next

Fig. 4. Analysis of the stability of WT and E164L at 60 °C. (a) The value of rootmean-square fluctuation (RMSF) for each residue in the simulation time of 50 ns. (b) The value of root-mean-square deviation (RMSD) as a function of simulation time.

data analysis. As indicated in Fig. 5, the Ala155-Ala166 region was very close to the Gly228-Leu231 region linked to the Ile240-Asn253 region by eight residues. Thus the stability of Ile240-Asn253 was affected by the mutation Glu164Leu in the Ala155-Ala166 region which influenced the Gly228-Leu231 region. Importantly, the residues of Ile240-Asn253 were very close to the active site of MTG. Therefore, these phenomena suggested that the changes of the amino acid type in residue 164 affected the sequence of residues in the loop region around the active site of E164L, leading to a certain effect on the binding of E164L to the substrate CBZ-Gln-Gly. Then, the interactions between the two regions of Ala155-Ala166 and Gly228-Leu231 were first studied. As shown in Fig. 6(a) and (b), when the type of residue 164 was changed from an acidic amino acid (Glu) to a hydrophobic amino acid (Leu), it was observed that, compared with WT, the secondary structure of the Asp159-Leu164 residues 6

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Fig. 5. Typical conformation of E164L at 50 °C at the end of MD simulation. The active sites Cys64, Asp255, and His274 were shown in green. The regions of Ala155-Ala166, Gly228-Leu231, Ile240-Asn253, and His277-Met288 were shown in red. The structure of E164L was colored in blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

of E164L changed from an initial α-helix to a random coil, which could explain, to some extent, why the RMSF value of the Asp159-Leu164 of E164L was higher than that of WT (Fig. 4a). This might cause the disappearance of the hydrogen bond between Asp159 and the main chain and side chain of Thr229 in E164L, and further lead to the larger distance between the region of residues 159–164 and that of residues 228–231. Compared with WT, for example, the number of contacts between Asp159-Glu164 and Gly228-Leu231 was more than 15 at the last 25 ns, while E164L displayed a low contact number below 3 at the whole simulation time (Fig. 6c). Meanwhile, the hydrogen bonds between Asp159-Glu164 and Gly228-Leu231 of WT were more than those of E164L (Fig. 6d). As for E164L, there was no hydrogen bonds between Asp159-Glu164 and Gly228-Leu231 during the whole MD simulation. These data indicated that the mutation Glu164Leu reduced both the atomic-atomic contacts and hydrogen bonds between Asp159-Glu164 and Gly228-Leu231 in E164L, resulting in the weaker interactions between Asp159-Glu164 and Gly228-Leu231 of E164L than WT. Therefore, the enhanced instability of Ile240-Asn253 linked to Gly228Leu231 by eight residues in E164L might be due to the weaker interactions between Asp159-Glu164 and Gly228-Leu231. Next, the interactions between the other two regions of Ile240Asn253 and His277-Met288 were further studied. The residues Ile240Asn253 was just located in one of the two loop regions close to the active site of MTG (the other loop region was His277-Met288). Thus, the interactions between the loop region 1 (Ile240-Asn253) and the loop region 2 (His277-Met288) were shown in Fig. 7, and the number of residue contacts within the 3.5 Å spacing in the two regions were shown in Supplementary Fig. 5, respectively. As indicated in Fig. 7a, there was a long distance between Ile240-Asn253 and His277-Met288, allowing the substrate CBZ-Gln-Gly to easily access to the active site of MTG and undergo a catalytic reaction. Compared with E164L, the space between two loop regions of WT became smaller at the end of MD simulation (Fig. 7c). As for E164L, the distance between two loop regions remained unchanged (Fig. 7b). Moreover, as shown in Supplementary Fig. 5, it could be seen that the number of contacts between the two loop regions of WT rapidly increased after 25 ns, but little contacts between the two regions of E164L was observed. These results indicated that CBZ-GlnGly was difficult to access to the active site of WT because of the smaller space of two loop regions at 50 °C, but CBZ-Gln-Gly was easier to pass

Fig. 6. Interactions of residues 228–231 and 159–164 in WT and E164L at 50 °C. (a) Interaction conformation of residues 228–231 and 159–164 of WT at 50 °C. (b) Interaction conformation of residues 228–231 and 159–164 of E164L at 50 °C. The hydrogen bonds were indicated by the blue dotted lines. The structure of WT was colored in pink. The structure of E164 L was colored in blue. (c) The atomic-atomic contacts number of residues 228–231 and residues 159–164 within a distance of 3.5 Å. (d) The number of hydrogen bonds of residues 228–231 and residues 159–164. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

the two loop regions to contact the active site of E164L in comparison to WT. The reason might be that the interactions of Ile240-Asn253 and His277-Met288 were significantly weak in E164L, because the increased instability of Ile240-Asn253 caused the enhancement of structure-flexibility for Ile240-Asn253 in comparison to WT at 50 °C. Therefore, it was beneficial to improving the contact, interaction, and reaction of substrate molecule with the active site of E164L. The cluster analysis of the two systems was shown in Fig. 8 to verify the accuracy of the trajectory analysis above and demonstrate the main representative conformations of WT and E164L during MD simulations of 50 ns. The conformational distribution of E164 L was more concentrated, and the cluster1 and cluster2 occupied 70% of the total conformation (Fig. 8b), indicating that E164 L had a more stable structure in comparison to WT. In the representative conformations of cluster1 and cluster4 in WT, the substrate CBZ-Gln-Gly has left the active site, but it was not far from the surface of MTG and bound by the loop region Ile240–253 and His277Met288 (Fig. 8a). This phenomenon was particularly obvious in the representative conformation of the fourth cluster. In the representative conformation of all clusters of E164 L, the substrate CBZ-Gln-Gly never

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Fig. 7. Interactions between loop region 1 and 2 at 50 °C. Loop region 1 including residues 240–253 and loop region 2 including residues 277–288 and the position of the small molecule substrate were exhibited in the figure. (a) Initial structure of MTG. (b) Typical structure of E164 L at the end of MD simulation. (c) Typical structure of WT at the end of MD simulation. The two loop regions were shown in purple. The structures of WT and E164L were colored in gray. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

The distance between the substrate molecule and the active site of MTG was further studied. It was reported that the Sulphur atom of Cys64 in MTG attacked the carbon atom in the CBZ-Gln-Gly amide in the first step of catalytic reaction, and then nucleophilic substitution was followed (Fig. 9a) [48]. Thus, the distance between the sulphur atom of the residue Cys64 of MTG and the carbon atom of amide group of the substrate of CBZ-Gln-Gly was used to represent the enzymatic activity. The distance between the sulphur atom of the residue Cys64 of both WT and E164L and the carbon atom of amide group of CBZ-GlnGly was calculated and shown in Fig. 9b. As for WT, it was clear that the distance between the sulphur atom

left the active site, and the loop regions Ile240–253 and His277-Met288 did not influence the free movement of the small molecule. At the end of the catalytic reaction, the reaction product could also quickly leave the active site without affecting the contact of the next substrate molecule with the active site. The representative conformations of the first and fourth clusters of WT appeared at the end of the MD simulation (Fig. 7c). It meant that WT would continue maintaining this complex conformation with substrate, which was not conducive to the catalytic reaction. Therefore, it was believed that the trajectory analysis and cluster analysis could clarify, to some extent, the mechanism of higher catalytic activity of E164L than that of WT.

Fig. 8. The representative scale arrangement conformations of WT (a) and E164 L (b) obtained by cluster analysis of the trajectories. 8

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Acknowledgements This work was supported by the National Natural Science Fund of China (31671806), the China Postdoctoral Science Foundation (2018M641660), the Open Project Program of State Key Laboratory of Food Nutrition and Safety (SKLFNS-KF-201813), and the Tianjin Natural Science Fund (17JCYBJC23700). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.bej.2019.107333. References [1] S. Beninati, M. Piacentini, The transglutaminase family: an overview: minireview article, Amino Acids 26 (2004) 367–372, https://doi.org/10.1007/s00726-0040091-7. [2] K. Yokoyama, N. Nio, Y. Kikuchi, Properties and applications of microbial transglutaminase, Appl. Microbiol. Biot. 64 (2004) 447–454, https://doi.org/10.1007/ s00253-003-1539-5. [3] I.M. Martins, M. Matos, R. Costa, F. Silva, A. Pascoal, L.M. Estevinho, A.B. Choupina, Transglutaminases: recent achievements and new sources, Appl. Microbiol. 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Fig. 9. Binding analysis of key residues of MTG to key chemical groups of the substrate in the catalytic reaction. (a) Binding conformation between active center of MTG and substrate CBZ-Gln-Gly. (b) Distance between S atom (Cys64) and C atom (amide group of CBZ-Gln-Gly) in WT and E164 L. It indicated the function of time for the simulations performed at 40 °C, 50 °C, and 60 °C.

of the residues Cys64 and the carbon atom of amide group of CBZ-GlnGly increased gradually with the increasing temperature during 25–50 ns of MD simulation (Fig. 9b). Especially for 60 °C, the distance was increased rapidly from initial 9 Å to about 40 Å, and then fluctuated around 36 Å, indicating that the substrate was moved away from the active site. Therefore, the activity of WT would be destroyed greatly at 60 °C. However, for E164L, the distance between the above two atoms remained stable during the whole simulation time regardless of the change of temperature, exhibiting that the complex structure of E164L and the substrate of CBZ-Gln-Gly kept stable in the selected temperature. The data of MD simulations showed that the mutation Glu164Leu indeed affected the interaction between substrate molecule and the active site of E164L, resulting in the improved catalytic activity of E164 L to CBZ-Gln-Gly in comparison to WT.

4. Conclusions In this study, a novel MTG mutant E164L with the enhanced activity and thermostability was obtained from a mutant library constructed by error-prone PCR. MD simulation studies indicated that mutations Glu164Leu weakened the interactions between the two loop regions adjacent to the active site and consequently increased the contact of substrate molecule to the active site in E164L. Generally, this work could improve the understanding of structure-activity relationship in MTG adapted to high temperature and provide theoretical foundation and preliminary information on the improved catalytic activity of MTG to meet the industrial requirements by protein engineering.

Authors’ contributions FFL and FPL conceived and designed the experiments. YHL, LH, MYS and JCS performed the experiments. YHL, YZL, LGJ, NW, SW, and SLS analyzed and interpreted the data. YHL wrote the manuscript. All authors read and approved the final manuscript. Declaration of Competing Interest The authors declare that they have no competing interests. 9

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