Remarkable Improvement of Methylglyoxal Synthase ... - Springer Link

Appl Biochem Biotechnol (2014) 172:157–167 DOI 10.1007/s12010-013-0404-y

Remarkable Improvement of Methylglyoxal Synthase Thermostability by His–His Interaction Malihe Mohammadi & Mona Atabakhshi Kashi & Shekufeh Zareian & Manoochehr Mirshahi & Khosro Khajeh

Received: 27 February 2013 / Accepted: 17 July 2013 / Published online: 21 September 2013 # Springer Science+Business Media New York 2013

Abstract Lately it has been proposed that interaction between two positively charged side chains can stabilize the folded state of proteins. To further explore this point, we studied the effect of histidine–histidine interactions on thermostability of methylglyoxal synthase from Thermus sp. GH5 (TMGS). The crystal structure of TMGS revealed that His23, Arg22, and Phe19 are in close distance and form a surface loop. Here, two modified enzymes were produced by site-directed mutagenesis (SDM); one of them, one histidine (TMGS-HHO), and another two histidines (TMGS-HHHO) were inserted between Arg22 and His23 (HO). In comparison with the wild type, TMGS-HHO thermostability increased remarkably, whereas TMGS-HHHO was very unstable. To explore the role of His23 in the observed phenomenon, the original His23 in TMGS-HHHO was replaced with Ala (TMGS-HHA). Our data showed that the half-life of TMGS-HHA decreased in relation to the wild type. However, its half-life increased in comparison with TMGS-HHHO. These results demonstrated that histidine– histidine interactions at position 23 in TMGS-HHO probably have the main role in TMGS thermostability. Keywords Histidine–histidine interaction . Thermostability . Methylglyoxal synthase . TMGS . Allosteric enzyme Abbreviation TMGS HO TMGS-HHO and TMGS-HHHO DHAP SDM

Thermus sp. GH5 methylglyoxal synthase TMGS original histidine Mutant TMGS Dihydroxyacetone phosphate Site-directed mutagenesis

M. Mohammadi : M. A. Kashi : M. Mirshahi : K. Khajeh (*) Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 14115-175, Tehran, Iran e-mail: [email protected] S. Zareian Department of Biological Sciences, Institute for Advanced Studies in Basic Sciences, Zanjan, Iran

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Appl Biochem Biotechnol (2014) 172:157–167

Introduction Interactions between amino acid side chains play a major role in determining the structure and function of proteins in aqueous solutions. For charged residues, attractive interactions are typically considered to be established only between a positive and a negatively charged side chain, leading to the formation of a salt bridge [1]. Salt bridges are now believed to be a stabilizing factor in the native state of proteins [2]. However, while unusual, close contacts have also been observed computationally between groups of residues with the same charge in peptides and proteins [3]. Recently, the study of interactions between like-charged ions, in particular between two positively charged moieties in solution, has gained a lot of attention among researchers [2]. This phenomenon was observed for the first time for arginine–arginine pairing [4, 5]. Then it was also demonstrated that histidine–histidine and histidine–arginine dipeptides could be stabilized in water [1]. Lately, in our laboratory a histidine–histidine pair in the Ca-lll binding site of α-amylase (BAA) was created by site-directed mutagenesis that show improved thermal stability [6, 7]. Therefore, we have created a histidine–histidine pair by histidine(s) insertion in methylglyoxal synthase gene. Methylglyoxal synthase (MGS) is an allosteric homohexameric enzyme that catalyzes an elimination reaction that converts dihydroxyacetone phosphate (DHAP) to orthophosphate and methyglyoxal (MG) in the first step of the methylglyoxal bypass in the Embden–Myerhoff pathway (glycolysis) [8]. MG is converted either to D-lactate via the glyoxalase system or to 1,2-propandiol (a commercial commodity) by glycerol dehydrogenase and aldehyde reductase which the latter is of industrial importance [9, 10]. Therefore, the study and thermostabilization of this enzyme is of interest. The MGS enzyme of many different species has been studied [10–13], particularly from Escherichia coli [14–16]. Recently, a gene encoding MGS from Thermus sp. GH5 (TMGS) was cloned, expressed [17], and its protein structure was studied by X-ray crystallography (PDB code 2XW6) (A. Shahsavar et al. unpublished work). Previous studies showed that TMGS gene was composed of 399 bp which encoded a polypeptide of 132 amino acids with a molecular mass of 14.3 kDa [17]. The crystal structure of TMGS revealed that His23, Arg22, and Phe19 are in close and form an appropriate loop for histidine insertion. In this survey, two modified enzymes were produced via site-directed mutagenesis. In order to study the role of histidine– histidine interaction on the TMGS, one and two histidines were inserted between Arg22 and His23. Then the stability, half-lives, and irreversible thermoinactivation parameters were analyzed and compared with that of wild type.

Materials and Methods Homology Modeling and Mutation Design The structure of TMGS had been previously modeled by our group (PDB code 2XW6). For the modeling procedure, the sequences of TMGS variants were submitted to the Swiss Model (http://swissmodel.expasy.org//SWISS-MODWL.html) and then the ribbon presentations were analyzed by PyMOL Viewer. Chemicals Pfu DNA polymerase, dNTP, and DpnI were purchased from Fermentas Life Science (Vilnius, Lithuania). Oligonucleotides were synthesized by Macrogen Inc. (Korea). The growth media and reagents were purchased from Liofilchem (Roseto degli Abruzzi, Italy).


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Dihydroxyacetone phosphate was purchased from Sigma-Aldrich (USA). 2,4-Dinitrophenyl hydrazine and other chemicals were obtained from Merck (Darmstadt, Germany). Site-Directed Mutagenesis Mutagenesis was carried out using the Quik-Change site-directed mutagenesis protocol described by Fisher and Pei [18]. The primers used were as follows (the codons inserted or replaced are underlined): TMGS-HHO TMGS-HHO TMGS-HHHO TMGS-HHHO TMGS-HHA TMGS-HHA

forward: 5′-CCTTTTGCCAGCGGCACCACCGGGAGGTCCTTG-3′ reverse: 5′-CAAGGACCTCCCGGTGGTGCCGCTGGCAAAAGG-3′ forward: 5′-CCTTTTGCCAGCGGCACCACCACCGGGAGGTCC-3′ reverse: 5′-GGACCTCCCGGTGGTGGTGCCGCTGGCAAAAGG-3′ forward: 5′-CCTTTTGCCAGCGGCACCACGCTCGGGAGGTCC-3′ reverse: 5′-GGACCTCCCGAGCGTGGTGCCGCTGGCAAAAGG-3′

Plasmid pET-21a (+) containing the TMGS gene was used as the template. The PCR reaction was carried out in a 50-μl volume containing 10 ng DNA template, 10× PCR buffer, 0.2 mM of each dNTP, 0.8 μM of each primer, and Pfu DNA polymerase (1.25 U). The mixture was heated at 95 °C for 5 min and then subjected to 22 cycles of thermal cycling at 95 °C for 1 min, 65 °C for 1 min, and 68 °C for 10 min. Then the PCR product was incubated with DpnI at 37 °C for 12 h and transformed to E. coli DH5-α [19]. Three colonies were randomly selected and confirmed by sequencing. For expression, plasmids were then transformed into E. coli BL-21 using calcium chloride transformation method. Expression and Purification of Wild-Type and Mutant Enzymes E. coli BL-21 cells harboring each of recombinant plasmids were grown overnight at 37 °C in 5 ml Luria–Bertani (LB) medium supplemented with ampicillin (100 μg/ml). An overnight culture was inoculated into fresh 500 ml culture medium (1% inoculation) containing ampicillin (100 μg/ml) and incubated at 37 °C until OD600 reached 0.5–0.6. IPTG (1 mM) was added to the culture medium at mid-log phase. Subsequently, the temperature was lowered from 37 to 30 °C which is suitable for production of adequate amount of protein. After 19 h, cells were harvested by centrifugation at 824×g for 20 min and resuspended in lysis buffer containing 50 mM imidazole (pH 6.2) and 1 mM phenylmethylsulfonyl fluoride. After sonication and centrifugation, the supernatant underwent heat shock at 70 °C for 15 min, and the precipitated proteins were removed by centrifugation at 2,500×g for 20 min at 4 °C. The resulted supernatant was dialyzed against 20 mM Tris buffer, pH 8.0, and applied onto Q-Sepharose column (15 cm×1 cm) equilibrated with the same buffer. Proteins were eluted with a linear gradient of NaCl (0–1 M) prepared in 20 mM Tris buffer (pH 8.0). The flow rate was set at 3 ml/min, and fractions containing MGS activity were collected. All the purification steps were performed at 4 °C. The purity of proteins was confirmed by SDS– PAGE according to the method of Laemmli [20]. Protein concentration was measured by Bradford method [21] using bovine serum albumin as standard. Enzyme Assay and Kinetic Characterization The enzyme activity was measured based on the procedure described by Hopper and Cooper [22, 23]. Briefly, 125 μl of 50 mM imidazole buffer (pH 6.2), 10 μl DHAP (15.6 mM), and 10 μl (16.8 ng) of the enzyme were incubated at 60 °C for 5 min. Then 0.1 ml of the mixture

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was added to 0.33 ml of 2,4-dinitro-phenyhydrazine reagent (0.1 % 2,4-dinitro-phenyhydrazine in 2 mM HCl) and then mixed with 0.9 ml water. After incubation at 30 °C for 15 min, 1.67 ml NaOH (10 % w/v) was added. After 15 min of incubation, the absorbance was measured at 550 nm. A molar extinction coefficient of 4.48×104 M−1 cm−1 was used to calculate the methyglyoxal concentration. Kinetic parameters measurements were carried out using different substrate concentrations (1–10 mM). Different concentrations of DHAP were used as the blanks in order to determine the activity. Steady-state kinetic parameters in the presence and absence of phosphate were fitted to Michaelis–Menten equation. Hill coefficient was calculated from the following equation: h . i ð1Þ Log v ðV max −vÞ ¼ nH log½S –log ðK 0 Þ where v and Vmax are velocity and maximal velocity of the enzyme, and nH is the Hill coefficient. K′ is related to Km but also contains terms related to the effect of substrate occupancy at one site on the substrate affinity of the other sites. According to this equation, the value of nH can be calculated by plotting log [v/(Vmax − v)] against log[S]. Determination of Thermal Stability Thermal stability was investigated by incubating the purified enzyme (at final concentration of 20 ng/ml) in 50 mM imidazole, pH 6.2, at 60, 70, 75, 80, 85, and 90 °C for different times. At regular intervals, samples were removed, cooled on ice, and the remaining activity was determined. Activity of the enzyme solution kept on ice was considered as a control (100 % activity). The half-lives (t1/2) of enzymes at given temperature were calculated. Results presented here are the mean from at least three repeated experiments in a typical run to confirm reproducibility. Calculation of Irreversible Thermoinactivation Parameters Enzyme inactivation can often be described by a first-order kinetic model. Therefore, plots of log residual activity versus time are linear under these conditions (data not shown). The rate constant (kinac) is used to calculate the activation energy according to the Arrhenius Eq. (2): k inac ¼ A e–Ea =RT

ð2Þ

where kinac (s−1) is the rate constant of inactivation process at temperature T (K); A, a preexponential factor; R, the gas constant (8.314 J mol−1 K−1); and Ea, the activation energy of the reaction. Therefore, the slope of the Arrhenius plot (−Ea/RT) was used to obtain Ea. The thermoinactivation parameters were calculated as follows:

ΔH # ¼ Ea −RT

ð3Þ

ΔG# ¼ RT lnðK B T =hÞ−RT lnk inac

ð4Þ

ΔS # ¼ ΔH # −ΔG# =T

ð5Þ


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Fig. 1 a View of the local environment surrounding Arg22 and His23 in wild-type TMGS (PDB code 2XW6). b Ribbon representation of this region in wild-type TMGS. Arg22 and His23 are in the surface loop

where KB is the Boltzmann constant (1.3805×10−23 J K−1); h, Planck’s constant (6.6256×10−34 J s); kinac (s−1), the rate constant of inactivation process; and T, the temperature (K). All measurements were performed in at least three independent experiments. Circular Dichroism Studies Far-UV spectra (190–260 nm) were recorded on a Jasco spectropolarimeter J-715 (Tokyo, Japan) using 1-mm path length quartz cell at the protein concentration of 0.2 mg/ml in 20 mM Tris buffer (pH 8.0). Results are presented as molar ellipticity [θ] (deg cm2 dmol−1), based on a mean amino acid residue weight of 108 for TMGS. The molar ellipticity [θ] was calculated from the formula [θ]λ=(θ×100MWR)/(cl), where c is the protein concentration (mg/ml), l the light path length in centimeters, and θ is the measured ellipticity in degrees at wavelength λ. Fluorescence Analysis Fluorescence measurements were carried out using a Perkin Elmer luminescence spectrometer LS 50B. Samples were excited at 280 nm and the emission spectra were recorded between 300 and 400 nm. All experiments were carried out at 25 °C and protein concentrations were 0.02 mg/ml in 20 mM Tris buffer (pH 8.0).

Results and Discussion Homology Modeling and Mutation Design To make minimal changes in the protein sequence and structure, the new introduced histidine(s) were decided to be near the original TMGS histidine residue, in a surface loop

Fig. 2 Amino acid sequence alignment of target region in TMGS with two modified enzymes. Original amino acids Arg22 and His23 (HO) are shown in bold and inserted histidines are underlined

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Fig. 3 Time course of thermal inactivation of wild type (filled circles), TMGS-HHO (filled squares), and TMGS-HHHO (filled triangles) at 75 °C (a), 80 °C (b), and 85 °C (c). Experiments were performed at least in triplicate and the standard deviations were within ±5 % of the experimental values

which does not interfere with the active site. The crystal structure of TMGS and spdv analysis (Swiss-PdbViewer) revealed that among the three existent histidines in TMGS, only His 23 was originally placed in a surface loop. Therefore, this loop was selected for histidine insertion (Fig. 1). This loop contains His23, Arg22, and Phe19. PyMOL Viewer indicated that introducing one or two histidine(s) between Arg22 and His23 does not disturb the basic enzyme structure as mentioned above. Therefore, two variants were produced by inserting one and two histidines separately between Arg22 and His23 (Fig. 2). Thermal Stability of Wild-Type and Mutated TMGS Irreversible thermoinactivation of purified wild-type and mutated enzymes was determined in 50 mM imidazole buffer (pH 6.2) at 60, 70, 75, 80, 85, and 90 °C (Fig. 3). After heat treatment for 120 min at 60 °C, wild type and TMGS-HHO retained nearly 100 % of their initial activity. But after 100 min of incubation in this condition, TMGS-HHHO lost nearly 40 % of its initial activity. The wild type and TMGS-HHO remained stable after 180 min of incubation at 70 °C, whereas TMGS-HHHO retained only 10% of its initial activity after 30 min (data not shown). The wild-type enzyme retained 50 % of its initial activity at 75, 80, and 85 °C for nearly 301, 25, and 5 min, respectively (Table 1). In comparison with the native enzyme, the half-life (t1/2) of TMGS-HHO increased significantly and was calculated to be 1,386, 247, and 182 min at 75, 80, and 85 °C, respectively. However, the thermostability of TMGS-HHHO, in relation to the wild type and TMGS-HHO, decreased severely. This mutant retained 50 % of its initial activity in these temperatures for 2.9, 2.8, and 2.1 min, respectively. Results revealed that addition of a single histidine next to His23 in TMGS-HHO enhanced its thermal stability compared to the wild type. Insertion of two histidines in the same site strongly decreased the thermal stability of TMGS. At 75 °C (optimum temperature of TMGS) [21], the half-life (t1/2) in TMGS-HHO increased about 4.6-fold and its value was approximately 100-fold, lower than the wild type in TMGS-HHHO. Table 1 Half-lives of TMGS and two mutant enzymes

Enzyme

Half-lives (min) 75 °C

80 °C

85 °C

TMGS TMGS-HHO

301.3±15.06 1386.2±69.31

25.2±1.26 247.2±12.36

4.5±0.22 182.1±9.10

TMGS-HHHO

2.9±0.14

2.8±0.14

2.1±0.10

90.1±7.2

81.1±5.7

38.2±2.3

TMGS-HHO

TMGS-HHHO

Ea

TMGS

Enzyme

37.5±2.2

80.4±4.0

89.4±6.2

21.1±1.3

25.2±1.8

24.2±1.4 16.4±1.1

55.2±3.3

65.2±4.6 37.5±1.9

80.4±5.6

89.4±5.4

ΔH#

TΔS#

ΔH#

ΔG#

80 °C

75 °C

Inactivation (kcal/mol)

Table 2 Thermodynamic parameters of wild type and mutant TMGS

21.3±1.7

24.6±1.5

22.8±1.6

ΔG#

16.2±1.0

55.8±3.9

66.6±3.3

TΔS#

38.2±2.7

80.4±5.6

89.4±6.2

ΔH#

85 °C

21.4±1.3

24.5±1.5

22.1±1.5

ΔG#

16.8±1.0

55.9±3.9

67.3±4.4

TΔS#

Appl Biochem Biotechnol (2014) 172:157–167 163

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Table 3 Kinetic parameters of TMGS and two mutant enzymes

a

Hill coefficient is for 1.5 mm concentration of phosphate


Enzyme

Km (mM)

kcat (s-1)

kcat/Km

nHa

TMGS

0.83±0.06

230±20.7

275.4

2.20

TMG-HHO TMGS-HHHO

0.67±0.05 1.45±0.1

153±10.7 138±11.0

227.3 94.8

1.58 2.12

In order to identify the role of His 23 in thermostability and instability of TMGS-HHO and TMGS-HHHO, a new single mutant was designed. For this reason, His23 (HO) in TMGS-HHHO was replaced with alanine (TMGS-HHA mutant). The time course inactivation of this mutant was subsequently determined at 75 and 80 °C. The half-life (t1/2) of this mutant at 75 and 80 °C was decreased by almost 136 and 8 min when compared to the wild type, and increased 162 and 14 min in comparison with TMGS-HHHO. Thermal stability and t1/2 analysis exhibited that the original histidine (His23) is an important determinant for TMGS-HHO thermostability. The thermostability of TMGS and its variants were in the following order: TMGS-HHO>wild type>TMGS-HHA>TMGS-HHHO. Thermal Inactivation Parameters After studying the thermostability of TMGS and its variants between 60 and 90 °C, the temperature dependence of the rate constant for inactivation was determined according to Arrhenius plot (as described in “Materials and Methods”). A simple scheme describing irreversible thermoinactivation of a protein would consist of two steps [24]: reversible denaturation step (D) followed by kinetically irreversible steps which lead to inactivation (I) of protein, depicted as: N ↔D→I Thermodynamic parameter for inactivation (Table 2) of TMGS-HHO shows that the energy barrier for inactivation (ΔG#) is the highest. Any factor which increased ΔG# would stabilize a protein [25]. It seems that a new histidine–histidine interaction in TMGS-HHO results in an increase in ΔG# as compared to the wild type. Meanwhile, Ea, ΔH#, and ΔS# parameters for TMGS-HHHO decreased noticeably, relative to wild type. These results are in agreement with the experimental observations for TMGS-HHHO.

Fig. 4 Far-UV CD spectra of wild type and mutants: 1 TMGS-HHHO, 2 wild type, 3 TMGS-HHA, 4 TMGS-HHO. The experiments were performed in triplicate


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Fig. 5 Intrinsic fluorescence spectra of 1 TMGS-HHO, 2 wild type, 3 TMGS-HHA, and 4 TMGS-HHHO at 280 nm. The experiments were performed in triplicate

It has been suggested that an inverse correlation exists between loop length and thermostability of enzymes [26, 27]. In TMGS-HHHO, two histidines were inserted between the native Arg22 and His23, so that His23, Arg22, Phe19, and two new histidines form a surface loop. Therefore, in comparison with TMGS-HO and wild type, the loop length has increased. This could be the reason for the instability of TMGS-HHHO and a decrease in values of thermodynamic parameters. Enzyme Kinetics As shown in Table 3, kinetic constants of the wild-type and mutant enzymes were calculated by analyzing Michaelis–Menten plot using Prism software version 5.04 (available at www.graphpad.com). The kcat and kcat/Km values for the wild-type TMGS were determined to be 230 s−1 and 275, respectively. These two parameters were decreased for modified enzymes. Previously, it has been observed that an increase in thermal stability is usually accompanied by a decrease in catalytic activity. As a result, protein rigidity would reduce fluctuation at the active site [28, 29]. In the case of allosteric behavior, the Hill coefficient decreased in TMGS-HHO compared to the wild type. Scrutton et al. reported that an increase in nH is ensued by a decrease in the thermal stability of the mutant glutathione reductase [30]. The Hill coefficient for TMGS-HHHO is close to that of the wild type with a very small decrease in nH value (Table 3). Circular Dichroism Circular dichroism (CD) was used to study the changes in secondary structure of all variants. The far-UV spectra of TMGS showed a considerable increase in negative ellipticity upon one histidine insertions which indicates a higher secondary structure content in this mutant (Fig. 4). CD spectra of TMGS-HHHO showed lower values of ellipticity than the wild type. In the case of TMGS-HHA, a slight increase in negative ellipticity was observed compared with that of the wild type. Fluorescence Intensity In order to investigate the tertiary structure of TMGS and its mutants, fluorescence emission measurements were performed. Fluorescence is emitted by intrinsic fluorophores of proteins

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like tryptophan and tyrosine residues. Structural changes in protein may expose the internal tryptophan/tyrosine residues to aqueous environment or bury the accessible residues in the core of the protein. Therefore, it is possible to indirectly follow the conformational changes of the enzyme through its intrinsic fluorescence emission. An increase of fluorescence intensity has been observed for TMGS-HHO (Fig. 5), whereas fluorescence intensity for TMGS-HHHO and TMGS-HHA was decreased compared to the wild type. More structural compactness leads to higher fluorescence intensity. These findings may reflect the increment of local compactness and rigidity in the structure of TMGS-HHO. Taken together with thermal stability data, it is proposed that TMGS-HHO variant is not only more compact but also more stable compared to the wild-type enzyme. Conclusion In the present study, we analyzed the impact of histidine–histidine interaction on TMGS thermostability by histidine insertion in a surface loop. Thermal stability and thermal inactivation studies showed that an increase in the number of adjacent histidines do not necessarily result in strong interactions between these amino acids and does not always have a positive effect on thermal stability. In the case of TMGS-HHHO, it is suggested that increasing the loop size can obscure the effect of the mentioned His–His interaction on stability. Furthermore, four positively charged amino acids located (three histidines and one arginine) in the mentioned loop could strongly disturb the protein conformation. Although both TMGS-HHA and TMGS-HHO contained two histidines, however, TMGS-HHO demonstrated higher thermostability. Results revealed that the presence of His residue at position 23 probably has a critical role in thermostability of TMGS. TMGS is a small homohexameric protein; therefore, any change in its structure is amplified. Consequently, these changes are observed as considerable changes in the t1/2, negative ellipticity, and fluorescence spectra. Previously, in the case of α-amylase (BAA), it was shown that histidine–histidine pairs improved the thermal stability [6, 7]. Here, for the first time histidine–histidine interactions have been studied in a hexameric enzyme with allosteric behavior. Data showed that interactions between like-charged amino acid residues at appropriate site(s) can improve the protein’s thermal stability. It seems such like-charged pairs may play a positive role in protein stabilization. But further evidence and more detailed structural studies (i.e., crystallographic studies) are required. Acknowledgment The authors express their gratitude to the research council of Tarbiat Modares University for the financial support during the course of this project.

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