Characterization and structural analysis of a leucine aminopeptidase using site-directed mutagenesis

Abstract

Amp0279 (EC 3.4.11.24, GenBank: CP000817.1) is a Co²⁺-dependent leucine aminopeptidase from the Lysinibacillus sphaericus C3-41 genome. After analyses using molecular docking and spatial structure analysis, site-directed mutagenesis mutants were performed as Amp0279-R131E, Amp0279-R131H, Amp0279-R131A and Amp0279-E349D. The optimum pH of Amp0279-R131E was shifted from the original 8.5 to 7.5, and the overall electrostatic potential was shifted towards acidic. Compared with the original enzyme, the mutant proteins all gained better structural stability, especially the apparent melting temperature (T_m) of Amp0279-R131H increased from 41.8 to 45.5 °C. Morever, when protein was bound to the substrate, the T_m of Amp0279-R131E was increased by 7.3 °C and Amp0279-R131H increased by 5.4 °C, compared to the original enzyme. This is consistent with the results that the mutants acquired higher binding energies to the substrates, and an increase the hydrogen bonding force. In addition, the molecular docking of mutant and substrate revealed that the truncation of R131 contributes to the increase in the binding capacity of the substrate molecules to the active centre. In contrast, the presence of π-Cation interactions generated by R131 with the substrate has an important effect on the ability of Amp0279 to hydrolyse the substrate. This study demostrated that R131 is a key site for activity and stability, which is important in the future exploration of the functional structure of Amp0279.

Key points

For aminopeptidase Amp0279, R131 is a key site for activity and stability. The mutant of Amp0279-R131E shifted its optimal pH from 8.5 to 7.5, and the T_m value increased by 7.3 °C with binding the substrate, in comparison with Amp0279.

Introduction

Peptidases are enzymes that catalyse the degradation of relatively large peptide fragments. They are divided into two classes: endopeptidases and exopeptidases. The first category includes enzymes that cleave peptide chains within peptide chains internally. In contrast, enzymes in the second category are used to cleave amino acid residues at the ends of peptides, and include carboxypeptidases (an one amino acid residue at the C-terminal end of a free peptide) and aminopeptidases (an one amino acid residue at the N-terminal end of a free peptide) (Gonzales and Robert-Baudouy 1996). Aminopeptidases catalyse the cleavage of amino acids from the amino terminus of many proteins and peptides. They are widely distributed in bacteria, plants and mammalian tissues, and have been found in the cytoplasm and in subcellular organelles (Reiland et al. 2004; Gilboa et al. 2001). Aminopeptidases are enzymes that specifically catalyse the cleavage of amino acid residues at the N-terminal position of peptides and proteins (Gonzales and Robert-Baudouy 1996). Aminopeptidases can be divided into three main classes on the basis of the number of amino acids cleaved from the polypeptide chain, the specificity of the substrate and the sensitivity to various protease inhibitors. Aminopeptidases that hydrolyse the first peptide bond in a polypeptide chain to release a single amino acid residue are known as aminoacyl peptidases (EC 3.4.11); aminopeptidases that remove either a dipeptide or a tripeptide from a polypeptide chain are known as dipeptidyl- and tripeptidyl peptidases (EC 3.4.14). Another class of enzymes that act only on dipeptides or tripeptides are known as dipeptidyl peptidases (EC 3.4.15) and tripeptidyl peptidases (EC 3.4.14.4) (Nandan and Nampoothiri 2020). Among them, the aminopeptidases (EC 3.4.11) catalyse the release of mostly hydrophobic amino acid residues. They have important physiological roles, such as the protein maturation and degradation of protein, and the regulation of hormone levels. Moreover, these enzymes may play an important role in the food industry by altering the texture, bitterness and flavour of food (Sierra et al. 2017). Of these, metalloaminopeptidases are the largest group of aminopeptidases.

Amp0279 (EC 3.4.11.24) is a leucyl aminopeptidase that has the capacity to hydrolyse L-Leucine-p-nitroanilide (Leu-pNA). Amp0279 gene was predicted in L. sphaericus C3-41 have been heterologously expressed in Escherichia coli. BL21 and B. subtilis WB800N. It is a novel Co²⁺-dependent aminopeptidase belonging to the M29 family (Zhao et al. 2022). Co²⁺ is commonly used as a metal for in vitro enzymatic studies (Altmeyer et al. 2010). Cobalt at the optimal concentration (100 µM) significantly enhanced the activity of Amp0279. It demonstrated the highest activity at 50 °C and pH 8.0. The ever-increasing applications of microbial aminopeptidases have necessitated continuous research and development into novel aminopeptidases with improved properties. The characteristics of aminopeptidase, including its substrate specificity, optimum pH, thermostability, resistance to solvents, etc. could be improved through gene manipulations and by protein engineering (Nandan and Nampoothiri 2017).

In this study, molecular docking simulations of Amp0279 with its substrate molecule, L-leucine-4-nitroanilide, were performed to identify the key sites that play a role in its binding to the substrate. The key sites were mutated via targeted mutagenesis to facilitate the analysis of the binding site with the highest binding energy. The differences in the enzymatic properties and protein structure of each mutant were analysed and correlations were found, laying the foundation for the targeted evolution of this aminopeptidase.

Materials and methods

Strains, plasmids and growth conditions

C3-41 is a wild Lysinibacillus sphaericus strain isolated in China in 1987, and its genome has been sequenced (Hu et al. 2008). Amp0279, a leucine-specific aminopeptidase that can exhibit higher enzymatic activity when catalysed by Co²⁺ (Zhao et al. 2022). Escherichia coli BL21 (Transgen, Beijing, China) and Bacillus subtilis WB800N (the China Center for Type Culture Collection, China) (Zhao et al. 2022) were used for protein expression. E. coli and B. subtilis were cultured in Luria–Bertani (LB) medium (1% peptone, 0.5% yeast extract and 1% NaCl) at 37 °C. Ampicillin (100 µg/mL) and chloramphenicol (25 µg/mL) were added to the culture medium as antibiotics. Pre-existing B. subtilis WB800N–Amp0279 recombinant bacteria were cultured in an LB medium containing chloramphenicol (25 µg/mL) overnight (37 °C, 220 rpm), diluted to 1% with fresh LB medium to an OD600 of 0.6–0.8 and induced by the addition of 0.2 mM IPTG for 24 h (30 °C, 220 rpm). The complete coding sequence of Amp0279 was amplified from the B. subtilis WB800N–Amp0279 genome with the primers listed in Table 1. All enzymes were purchased from Takara Bio (Shiga, Japan). Cloning primers were synthesized by Tsingke Biotech (Wuhan, China). All chemical reagents were analytical grade.

Table 1 Primers relevant in the study

Molecular binding analysis of substrate binding sites and construction of the variants

The homology of Amp0279 was modelled using the website SWISS-MODEL (https://swissmodel.expasy.org/) (Figure S1). Amp0279 has the ability to hydrolyse L-leucine-4-nitroanilide. Docking between Amp0279 and L-leucine-4-nitroanilide was simulated by the software AutoDock, and the key docking sites were analysed (Figure S2). Amp0279 was targeted for mutation using PCR with the primers listed in Table 1.

Expression and purification of proteins

Linearisation of pET-28a(+) (Takara Bio, Shiga, Japan). by inverse PCR followed by recombination of gene fragments from Amp0279 and its mutants into pET-28a(+) by homologous recombination, and the resulting recombinant plasmids were transformed into E. coli DH5α for the modification of the recombinant plasmids and multiple cloning. The T7-tag and the 6×His-tag at the N-terminus in pET-28a(+) were excised by reverse PCR. The effect of excess tags on the protein structure was removed, and the His-tag at the C-terminus was retained for purification. The positive transformants were transferred into LB medium containing 30 µg/mL of kanamycin and incubated at 37 °C for 12 h. The recombinant plasmid was then transferred into E. coli BL21 for protein expression. E. coli BL21–Amp0279 was cultured in LB medium overnight (37 °C, 220 rpm), diluted to 1% with fresh LB medium to an OD600 of 0.6–0.8, and induced by adding 0.2 mM of IPTG for 24 h (30 °C, 220 rpm). After centrifugation at 8000 rpm for 10 min at 4 °C, the bacterial precipitate was resuspended in PBS, broken by ultrasound for 15 min and centrifuged (4 °C, 8000 rpm, 10 min). The supernatant was collected for analysis of the proteins. Amp0279 and its mutants were purified by Ni-NTA His Bind Resin (7sea biotech, Shanghai), and the purified solution was collected. Imidazole was removed from the eluate by a G25 gravity desalting column (Bersee, Beijing), and the eluate was collected for subsequent studies. The purified proteins were validated with SDS-PAGE protein gel for use in subsequent experiments, and the marker was the Prestained Protein Ladder (20350ES72, Yeasen, Shanghai).

Characterization of activity and stability

The activity of aminopeptidase was detected using the p-nitroaniline method (Tang et al. 2016). The reaction was carried out in a buffer containing 100 µmol/L Co²⁺ and 50 mM of 1 mM L-leucine-4-nitroanilide (Sigma-Aldrich, USA) as a substrate for 10 min, and the reaction was terminated by adding 40% glacial acetic acid. The absorbance was detected at OD405. The optimal reaction temperature was explored, and the activity of Amp0279 and its mutants was assayed at 30, 35, 40, 45, 50 and 55 °C. To test their thermostability, solutions of the protease were incubated at 30, 35, 40, 45, 50 and 55 °C for 1 h. To explore the optimum pH, reactions were carried out in the following buffers: 50 mM Na₂PO₄ and citric acid (pH 6.5, 7.0 or 7.5), 50 mM Tris-HCl (pH 7.5, 8.0, 8.5 or 9.0) and 50 mM glycine–NaOH (9.0, 9.5). To test their pH stability, the protease solutions were incubated in buffers with different pH values for 1 h and the activity of their enzymes was assessed. One enzyme unit was defined as the amount of the enzyme that hydrolysed 1.0 µmol of the L-leucine-4-nitroanilide substrate per minute. Configured L-leucine-4-nitroanilide solutions of different concentrations (0, 20, 40, 60, 80 mM) to calculate the K_m values of the enzyme and substrate. This serves as the basis for determining the affinity between enzymes and substrates. Measure the absorbance value at 405 nm for 3 min under the same reaction conditions. Draw a double reciprocal curve with the reciprocal of substrate concentration as the horizontal axis and the reciprocal of absorbance value as the vertical axis to obtain the K_m value of the enzyme.The absorbance values of 0, 0.05, 0.1, 0.15, 0.2 and 0.25 mM p-nitroaniline at OD405 were detected, and their standard curves were plotted. The specific activity of each protease was calculated by using BSA Standard Solution (Takara Bio, Japanese). The protein concentration was detected by the BSA method by taking 4 µL of 0, 25, 125, 250, 500, 750 and 1000 µg/mL of BSA Standard and mixing it with 200 µL of Bradford Dye Reagent, respectively, and then detecting its absorbance value at OD595 after reacting at room temperature for 5 min to plot the BSA standard curve. At the same time, 4 µL of the protein purification solution already obtained was taken and reacted with 200 µL of Bradford Dye Reagent to detect its absorbance value, and the concentration of individual proteins was calculated to obtain its specific enzyme activity. The 20 µL of purified protease was mixed with 5 µL of SYPRO™ Orange protein gel stain (invitrogen, US), and the apparent melting temperature (T_m) value of the protein was measured via differential scanning fluorimetry (DSF) using a CFX connect fluorescence quantitative PCR instrument (BIO-RAD, Singaporean) (Wang et al. 2022). Another set of additional 1 mM substrate was added for the determination of T_m values.

Results

Construction of the variants

The homology of Amp0279 was modelled using SWISS-MODEL (https://swissmodel.expasy.org/) using Staphylococcus aureus AmpS as a template (Figure S1). Molecular docking simulations were performed with the 3D structure of the L-leucine-4-nitroanilide substrate (Figure S2). According to the combined AutoDock and PyMOL analyses, among the three docking modes with the highest binding energies, in addition to the active site, amino acid 131 generated hydrogen bonding forces with the substrate molecule. At the same time, amino acid 131 produced a π-Cation Interaction with the substrate. In Figure S2, arginine 131 produced a π-Cation Interaction with a substrate molecule. π-Cation Interaction plays an important role in molecular recognition and binding. Finally, the arginine R of 131 was determined to be the mutation site (Figure S2). According to the acidity and hydrophilicity of the amino acids, E, H and A were selected to replace the original R. Site 349 was also mutated from Glutamate to Aspartate. Amp0279 was targeted by PCR to construct the mutants: R131E, R131H, R131A and E349D (Figure S3). They were all successfully expressed in E. coli BL21. Among the mutants, in terms of the spatial structure, Amp0279-R131A provided more space for the substrate molecules to enter the centre of the active site, followed by Amp0279-R131E and Amp0279-R131H (Figure S4). To explore this phenomenon, we performed molecular docking simulations with the substrate molecules for the mutants Amp0279-R131E, Amp0279-R131H and Amp0279-R131A. The molecular docking results showed that the individual mutants exhibited higher binding energy effects relative to the highest binding energy of Amp0279. At their active centres, the highest binding energies increased to − 8.24 Kcal/mol for Amp0279-R131E, − 7.44 Kcal/mol for Amp0279-R131H and − 7.59 Kcal/mol for Amp0279-R131A. Compared to the maximum binding energy of − 7.36 Kcal/mol for Amp0279, the substrate bound better to the mutated protein.

Characterization of activity and stability

In a follow-up study, we selected pET-28a(+) as the expression vector for Amp0279 and its mutants. Recombinant plasmids containing the His-tag were successfully constructed using homologous recombination, namely, Amp0279, Amp0279-R131E, Amp0279-R131H, Amp0279-R131A and Amp0279-E349D. After successful induction of expression in E. coli BL21, the target protein was successfully purified by Ni-NTA (Fig. 1). The five proteins were assayed for their activities at different temperatures (30, 35, 40, 45, 50 and 55 °C) and at different values of pH (6.5, 7.0, 7.5, 8.0, 8.5, 9.0 and 9.5). As seen in Fig. 2A, an optimum pH of 8.5 was detected for Amp0279 and Amp0279-E349D; the optimal pH for Amp0279-R131E is 7.5; the optimal pH for Amp0279-R131A and Amp0279-R131H is 8.0. Apparently, the optimum pH of the protein decreased to varying degrees as the amino acid at Site 131 changed from basic arginine to neutral alanine and acidic glutamate. As seen in Fig. 2B, the optimum temperature of Amp0279, Amp0279-R131E and Amp0279-R131H at pH 8.0 was 45 °C, whereas the optimum temperatures of Amp0279-R131A and Amp0279-E349D decreased to 35 and 40 °C, respectively. Afterwards, the protein content of the individual protease solutions was measured by the BSA method. Their specific activity was calculated under their optimal reaction conditions. Table 2 shows that the specific activity of Amp0279-R131E and Amp0279-R131H decreased significantly compared with the proenzyme, whereas the specific enzyme activity of Amp0279-E349D was slightly higher than that of Amp0279. Then draw a double reciprocal curve graph (Figure S5). The affinity results between enzymes and substrates showed a significant increase in the K_m values of Amp0279-R131E and Amp0279-R131A. The K_m value of the original enzyme is 2.70 moL/L, while the K_m values of Amp0279-R131E and Amp0279-R131A have increased to 330.66 moL/L and 20.86 moL/L. Indicating a decrease in affinity between the two and the substrate. However, the K_m of the mutant Amp0279-R131H showed no significant changes compared to the original enzyme. The possible reason may be due to changes in the acidity or alkalinity of the amino acids at the mutation site. Similarly, mutations to alkaline amino acids do not have a significant effect on the affinity of the enzyme to the substrate.

Fig. 1

SDS-PAGE analysis of Amp0279 and its mutants. Marker: Prestained Protein Ladder

Fig. 2

Assay of the enzymatic properties of Amp0279 and its mutants. A Detection of the optimum pH for the reaction. Reaction temperature: 45 °C. B Detection of the optimum reaction temperature. Reaction condition: pH 8.0. C Detection of pH stability. Reaction temperature: 45 °C. D Detection of thermal stability. Reaction condition: pH 8.0. Black, Amp0279; red, Amp0279-R131E; orange, Amp0279-R131H; green, Amp-R131A; blue, Amp0279-E349D

Table 2 Enzymatic properties of Amp0279 and its mutants

pH and temperature stability

Subsequently, Amp0279 and its mutants were incubated at different temperatures (30, 35, 40, 40, 45, 50 and 55 °C) and pH values (6.5, 7.0, 7.5, 8.0, 8.0, 8.5, 9.0 and 9.5) for 1 h, and the thermal stability and pH stability of the protease were tested. As seen in Fig. 2C, D, Amp0279-R131H and Amp0279-R131A exhibited higher and more stable enzyme activity in the pH 6.5–9.0 range. In contrast, Amp0279-R131E and Amp0279-E349D were less affected by pH in the range of 6.5–8.0 range. Overall, all four mutant proteins showed superior stability to the original enzyme.

Structural analysis based on Tm

Differential scanning fluorimetry (DSF) was used to detect the apparent melting temperature (T_m) values of the proteins. As shown in Fig. 3A, the T_m values of Amp0279, Amp0279-R131E, Amp0279-R131H, Amp0279-R131A and Amp0279-E349D were 41.8, 42.3, 45.5, 43.3, and 43.1 °C, respectively. The protein begins to undergo structural changes at a certain temperature. The specific folding structure of peptide chains is disrupted, leading to unfolding phenomenon. All mutants showed increased structural stability. The mutant Amp0279-R131H had a T_m value 3.7 °C higher than that of Amp0279. This indicated that the mutant Amp0279-R131H has a higher unchaining temperature and thus had greater structural stability. On the results of T_m measurements of the structures produced by the binding of the protein to the substrate, it was observed that the T_m values of Amp0279, Amp0279-R131E, Amp0279-R131H, and Amp0279-R131A were 36.9, 44.2, 42.3, and 40.4 °C, respectively. When the intermolecular binding energy is negative, proteins can bind to substrates under natural conditions. And it was confirmed in the early experimental process that protein and substrate can undergo binding catalytic reactions at room temperature. Therefore, it can be inferred that binding has occurred between the protein and substrate after mixing. The protein substrate complex as a whole is taken as the research object, and the protein concentration and substrate concentration are ensured to be consistent between each experimental group to ensure the accuracy of the experimental results. The results indicate that the mutant has high structural stability after binding to the protein substrate. This indicates that the structural stability is enhanced during the catalytic reaction process, especially Amp0279-R131E and Amp0279-R131H (Fig. 3B). This is consistent with the prediction of molecular binding energy. This suggests that the mutants possess higher structural stability for substrate binding in the hydrolysis reaction. After aligning Amp0279 with Amp0279-R131E, Amp0279-R131H, and Amp0279-R131A, the increased substrate-binding capacity of the active centre in the mutated proteins was analysed, as well as the increased structural stability of the active centre caused by the mutation, contributed to the change in the T_m value of the mutated proteins, especially Amp0279-R131E.

Fig. 3

T_m values of Amp0279 and its mutants determined by a differential scanning fluorescence assay (DSF). A No substrate. B Contains substrate

Discussion

The mutant Amp0279-R131E shifted its optimum pH from 8.5 to 7.5, and the mutant Amp0279-R131H increased its T_m value by 3.7 °C to give it better structural stability. Molecular docking was used to explore the effect of Arginine 131 on the protein’s properties by site-directed mutagenesis. Three different mutations were ultimately chosen to target the arginine at locus 131. The first choice was to replace it with histidine, which is also a basic hydrophilic amino acid. Next, glutamic acid, which is also hydrophilic, was substituted, the difference being that the amino acid was changed from basic to acidic. Finally, the neutral hydrophobic alanine was chosen to truncate the original amino acid in the spatial structure. Next, we analysed the resulting structures using PyMOL. Amp0279-R131E, Amp0279-R131H and Amp0279-R131A were analysed for protein contact potential (Fig. 4). After we mutated the basic amino acid at Site 131 to an acidic amino acid (i.e., arginine) to glutamic acid, the protein contact potential of the enzyme decreased from − 73.478 to − 74.857 Kcal/mol (Fig. 4B) (Li et al. 2019). The electrostatic forces on the surface shifted to negative values overall, and the proteins became biased towards acidity. Analysis of the physicochemical properties of the proteins using ProtParam (Expasy, ProtParam tool: https://web.expasy.org/protparam/) showed that the pI value of Amp0279-R131E decreased from 4.87 to 4.81 relative to Amp0279. Considering both the protein contact potential and pI values, we deduced that the mutation of arginine to glutamate at Site 131 was responsible for the decrease in the optimal pH of the mutant Amp0279-R131E from the original 8.5 to 7.5. The Grand average of hydrophilicity (GRAVY) is translated as the average coefficient of hydrophilicity. If this value is negative, it indicates that the protein is hydrophilic, otherwise it is hydrophobic. After the hydrophilic amino acid of Amp0279-R131A was changed to a hydrophobic amino acid. Its GRAVY value has also been changed from − 0.215 to − 0.199, indicating an increase in protein hydrophobicity.

Fig. 4

Protein contact potential of Amp0279 and its mutants. A Amp0279. B Amp0279-R131E. C Amp0279-R131H. Blue, basic; red, acidic

As shown in Fig. 5, after mutation of the amino acid in site 131, Amp0279-R131E, Amp0279-R131H and Amp0279-R131A all underwent structural truncation. In contrast to Amp0279, the mutants had a larger pocket in the active centre of the protein, theoretically providing more favourable conditions for the substrate molecules to enter deeper into the active centre (Figure S4). By analysing the spatial structure in the best docking results, we found that the higher the binding energy, the deeper the substrate molecules penetrated into the active centre in the structure in the following order: Amp0279 < Amp0279-R131H < Amp0279-R131A < Amp0279-R131E (Fig. 5). To test this theory, the Protein-Ligand Interaction Profiler (https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index) was adopted to analyse the binding of Amp0279 and its mutants to substrates that generate the force of action. The results showed that Amp0279 generated only 2 hydrogen bonds with the substrate molecule (Fig. 5A), while the complex of Amp0279-R131E with the substrate generated 4 hydrogen bonds (Fig. 5B), and the rest of Amp0279-R131H and Amp0279-R131A both generated 3 hydrogen bonds after docking with the substrate (Fig. 5C, D). The presence of Hydrogen Bond contributes to the tightness of inter-individual bonding. This phenomenon is highly consistent with the results of T_m values of protein-substrate bonds detected by DSF method. Validating the results produced by molecular docking, Amp0279-R131E possessed a higher docking binding energy (− 8.24 Kcal/mol), higher than that produced by Amp0279 (− 7.36 Kcal/mol). Meanwhile, in the substrate-bound state, Amp0279-R131E relative to Amp0279 T_m value was increased by 7.3 °C (Fig. 3B). The docking binding energy of Amp0279-R131H to the substrate has also changed from the original − 7.36 Kcal/mol to -7.44 Kcal/mol. This was followed by an increase in T_m value of 5.4 °C after Amp0279-R131H formed a complex with the substrate molecule. This result remains consistent with the hydrogen bonding analysis and docking binding energy effects. When the protein was in monomeric form, however, Amp0279-R131H gained higher structural stability with an increased T_m value of 3.7 °C (Fig. 3A). The enzyme reaction occurs at a temperature higher than the T_m value. At the optimal reaction temperature, enzymes have the best ability to catalyze substrate hydrolysis. The T_m value reflects the dissociation of protein structure at a certain temperature. The stability of protein structure is not directly related to the optimal reaction temperature. In contrast, Amp0279-R131E, Amp0279-R131H and Amp0279-R131A showed a significant decrease in their specific activity relative to the original enzyme (Table 2), which was caused by changes in the ability of the active centre in the structure of the mutated proteins to catalyse the substrate. At the same time, the K_m values of Amp0279-R131E and Amp0279-R131A increased. This indicates that both have weak binding abilities with substrates and display excessively high K_m values in the same detection environment. A higher substrate concentration is required to reach half of the maximum rate V_max. This means that at low substrate concentrations, the enzyme has insufficient affinity for the substrate and the reaction rate is slower. When the substrate concentration is high, the binding between the enzyme and the substrate will be enhanced, and the reaction rate will also increase, which needs further research. In enzyme-catalysed reactions, π-Cation Interaction stabilises the binding of substrate molecules, thereby facilitating the reaction. However, after mutation of site 131, all three mutants lost the π-Cation Interaction with the substrate (Fig. 5B, C). In protein-substrate interactions, π-Cation interaction occurs for molecules rich in positive charges (Dougherty 1996; Meyer et al. 2003). The π-Cation interaction comprises a substantial electrostatic characteristics, and it has a considerable effect on ligand binding (Mahadevi and Sastry 2013; Gebbie et al. 2017). Loss of π-Cation Interaction may be a factor in the decreased specific enzyme activity of Amp0279-R131E, Amp0279-R131H and Amp0279-R131A.

Fig. 5

Optimal docking positions of the substrate molecules in Amp0279 and its mutants. A Amp0279. B Amp0279-R131E. C Amp0279-R131H. D Amp0279-R131A. Red, amino acid at Position 131; blue, site of the active centre interacting with the substrate. Stick model of the substrate molecule, L-leucine-4-nitroanilide. Yellow dashed line, hydrogen bond. White dashed line, hydrophobic Interaction. Orange dashed line: π-Cation Interaction

In addition, structural analyses showed that Amp0279-R131E, Amp0279-R131H and Amp0279-R131A all lost the hydrogen-bonding force with E349 relative to the period before the mutation, while no new hydrogen-bonding force was created (Figure S6). This was mainly due to changes in the R side chain group of the amino acid. By analysing the spatial structure of the amino acids at Site 349, we found that the glutamate at this site in Amp0279 generated hydrogen-bonding forces with T376 and R131 (Fig. 6A). While T376 connected to H377 as the active centre site, E349 connected to Y351 as the active centre via A350. Thus, E349 had an effect on the sites of the two active centre in terms of the spatial structure. To investigate the interaction of glutamate at Site 349 with arginine at Site 131 on the active centre, we truncated the glutamate at Site 349 and replaced it with aspartic acid, both of which are acidic hydrophilic amino acids. The Amp0279-E349D mutated in this way similarly lost its hydrogen bond to Site 131, but still retained this hydrogen bond to T376 (Fig. 6B). A hydrogen bond for the interaction of D349 with A350 was added to the mutant Amp0279-E349D relative to Amp0279 (Fig. 6B). This hydrogen bond caused the folding angle of A350 to decrease from 124.9° to 124.1°, and the pull of D349 on T376 caused T376 to lose one of its hydrogen bonds to S374. The folding angle of the T376 increased from 121.1° to 122.2°. These changes in the two folding angles made Y351 and H377, which are the active sites, more compact in their spatial structure, and the distance between the two amino acids reduced from 3.4 to 3.3 Å. The tightening of the active centre structure may be the key to improving its catalytic ability, increasing the specific activity of Amp0279-E349D relative to Amp0279 to 121.91% of the original.

Fig. 6

Hydrogen bonding forces analysed for the mutation at Site 349 targeting Amp0279. A Amp0279. B Amp0279-E349D. Yellow dashed line, hydrogen-bonding forces generated between the amino acids; blue dashed line, distance between two atoms

Histidine affinity purification is a common method of purifying recombinantly expressed proteins. The effect of His-tag on protein structure and function is usually ignored during experiments. However, in some cases, the introduction of His-tags leads to changes in the proteins structure and may also interfere with the binding interactions (Thielges et al. 2011). For example, the addition of a His-tag changed the structure and sometimes the function of the target protein. At the same time, a His-tag can also lead to changes in the spatial structure of the active site of the protein (Chant et al. 2005), resulting in a decrease in the stability of the protein’s structure (Esteban-Torres et al. 2012); it can also affect the protein’s expression and solubility (Noirclerc-Savoye et al. 2015). Other comparative experiments yielded more significant data, where the recombinant vectors N-His-rXAn11 and N-C-His-rXAn11 were constructed at the N-terminal and C-terminal ends of xylanase, respectively, but the relative activity of N-His-rXAn11 increased by about 52% (Elgharbi et al. 2023). In this study, pET-28a(+) was chosen as a self-contained 6×His-tagged vector with 6× His-tags at both the N- and C-termini of its MCS region. In a preliminary study, we retained the 6×His-tags at the N-terminus on the pET-28a(+) vector. However, it failed to bind to the Ni-NTA purification column during subsequent purification. We believe that the possible reason was that the His-tag at the N-terminal end affected the folding of the original protein, and the 6×His-tag at the N-terminal end was not sufficiently exposed or functional during the folding of the protein. In this study, we added C-terminal His-tags for purification of the proteins. However, the influence of the C- and N-termini on the properties of proteins cannot be ignored, so it was important to find purification methods that did not rely on purification tags in subsequent studies. Among other methods of purifying aminopeptidases, graded ammonium sulphate precipitation is very efficient for PepA (EC 3.4.11.7), which has a purity of precipitation of more than 95% at very low concentrations of ammonium sulphate of 5% (Stressler et al. 2017).

In this study, we used molecular docking for Amp0279 and its substrate molecule, L-leucine-4-nitroanilide, to identify the key sites. The mutants Amp0279-R131E, Amp0279-R131H, Amp0279-R131A and Amp0279-E349D were obtained by mutating Locus 131 and Locus 349 by site-directed mutagenesis. One of them, Amp0279-R131E, produced a significant shift in the optimal reaction pH of the protein by changing the acidity of the amino acids. Amp0279-R131H improved the structural stability. Mutants have wider pH stability and better structural stability, which allows proteins to adapt to more industrial production situations. Amp0279-E349D has a significant effect in terms of improving the specific activity of the protein through the effect of the hydrogen-bonding force on the folding angle of the amino acids. The results of this study provide a basis for subsequent studies on the mechanism of action of Amp0279, improvements in enzyme activity and the directed evolution of Amp0279. An important role for site 131 in the hydrolysis of substrate molecules by Amp0279 was identified. The selection of expression vectors as well as the utilisation of soluble tags that can improve protein solubility are also possible directions for improving Amp0279 expression.