Characterization of Glutamate Decarboxylase from Lactobacillus

Nov 21, 2014 - Hee Seon Lim , Dong-Ho Seo , In-Tae Cha , Hyunjin Lee , Young-Do Nam , Myung-Ji Seo. World Journal of Microbiology and Biotechnology ...
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Characterization of Glutamate Decarboxylase from Lactobacillus plantarum and Its C‑Terminal Function for the pH Dependence of Activity Sun-Mi Shin,†,∥ Hana Kim,§,∥ Yunhye Joo,† Sang-Jae Lee,# Yong-Jik Lee,† Sang Jun Lee,⊥ and Dong-Woo Lee*,† †

School of Applied Biosciences, Kyungpook National University, Daegu 702-701, South Korea SAMSUNG BIOEPIS Corporation, Incheon 406-840, South Korea # Department of Bio-Food Materials, Silla University, Busan 617-736, South Korea ⊥ Infection and Immunity Research Center, Korea Research Institute Bioscience and Biotechnology (KRIBB), Daejeon 305-806, South Korea §

ABSTRACT: The gadB gene encoding glutamate decarboxylase (GAD) from Lactobacillus plantarum was cloned and expressed in Escherichia coli. The recombinant enzyme exhibited maximal activity at 40 °C and pH 5.0. The 3D model structure of L. plantarum GAD proposed that its C-terminal region (Ile454−Thr468) may play an important role in the pH dependence of catalysis. Accordingly, C-terminally truncated (Δ3 and Δ11 residues) mutants were generated and their enzyme activities compared with that of the wild-type enzyme at different pH values. Unlike the wild-type GAD, the mutants showed pronounced catalytic activity in a broad pH range of 4.0−8.0, suggesting that the C-terminal region is involved in the pH dependence of GAD activity. Therefore, this study may provide effective target regions for engineering pH dependence of GAD activity, thereby meeting industrial demands for the production of γ-aminobutyrate in a broad range of pH values. KEYWORDS: pH dependence, glutamate decarboxylase, C-terminal region, GABA, Lactobacillus plantarum

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INTRODUCTION Because γ-aminobutyrate (GABA) is a suppressive neurotransmitter present in the mammalian brain and spinal cord, it is used therapeutically for the treatment of headaches, tinnitus, and hypobulia and secondarily for stroke, head trauma, or cerebral arterial disorders, improving blood flow, oxygen supply, and metabolism in the brain.1−4 In addition, GABA has antioxidant, hypolipidemic, and anti-inflammatory effects, which can be used as an antiaging neutraceutical in the cosmetics and health-care food industries.5,6 Recently, milk products containing high contents of GABA as a food additive, which is produced by Lactobacillus fermentation, have been developed for lowering blood pressure.7 For these applications, immobilization of GABA-producing enzymes or strains derived from foodgrade lactic acid bacteria (LAB) or other microorganisms that are generally accepted as safe (GRAS) has been attempted to develop a biological process for the production of highly concentrated GABA.6,8−10 In fact, acid-tolerant microbial cells would be more practically efficient and feasible than enzymes for the production of GABA. Nevertheless, one may infer that engineered glutamate decarboxylase (GAD) with a slightly acidic pH optimum for catalytic activity can be compatible with immobilization conditions for GABA-producing bacteria, resulting in much higher yields of GABA production. Alternatively, immobilization of GAD would be also beneficial with respect to enzyme activity and stability at low pH conditions, where GAD has an optimum pH.11 GAD is a pyridoxal 5′-phosphate (PLP)-dependent intracellular enzyme, which catalyzes the irreversible α-decarboxylation of 12,13 L-glutamate to GABA. It has been noted that the GAD © XXXX American Chemical Society

system not only neutralizes acidic intracellular and extracellular environments but also converts cellular membrane potential depending on the external glutamate species by decarboxylating an acidic substrate (glutamate) into a neutral compound (GABA) via incorporation of H+.13−18 GABA can then be exported into the extracellular medium by the GadC protein, thereby contributing to local alkalization of the extracellular environment.19,20 Indeed, because most bacterial GADs with their acidic pH optima (pH 3.8−4.6) are expressed in response to environmental stress,13,21 those enzymes in glutamate metabolism can contribute to regulation of intracellular pH at acidic pH.14,21,22 Other acidinduced degradative amino acid decarboxylases include arginine (adiA), lysine (cadA), and ornithine (speF) decarboxylases.23−26 However, only amino acid decarboxylation and arginine deimination (ADI) systems provide acid protection, being optimally active at moderately acidic pH.22,27 Some bacteria such as Escherichia coli,28 Listeria monocytogenes,15,29 and Lactobacillus brevis30 have two GAD isoforms (GadA and GadB), which have very similar kinetic and physicochemical properties.31 In Escherichia coli, GadA and GadB have been identified as structural constituents of the GAD system13,15 and are known as the most potent acid resistance systems.32,33 However, the structural and molecular basis of GAD catalysis at low pH values remains unclear. Received: September 27, 2014 Revised: November 16, 2014 Accepted: November 21, 2014

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Purification of Recombinant L. plantarum GAD. Bacterial pellets were thawed and resuspended in 50 mL of 1× His-binding buffer (0.5 M NaCl, 20 mM Tris, 5 mM imidazole; pH 7.9) and disrupted by sonication. The lysate was centrifuged at 14000g for 20 min to remove cell debris, and the supernatant was loaded on a His-bind resin column (5 mL) equilibrated with the 1× His-binding buffer. The column was washed with 10 column volumes of wash buffer (0.5 M NaCl, 20 mM Tris, 60 mM imidazole, pH 7.9), and 250 mM imidazole was applied to elute the recombinant protein. The fractions containing enzyme activity were pooled and dialyzed against 10 mM sodium acetate buffer (pH 7.0). To remove the His-tag from the fusion protein, we added 7 U of thrombin (HTI, USA) to the dialyzed fusion proteins (∼10 mg) for 16 h to yield intact GAD without any 6× His tag. For further purification, the reaction mixture was subjected to a HiLoad 16/60 Superdex 200 gel filtration column (GE Healthcare, USA) pre-equilibrated with 50 mM Tris-HCl (pH 7.5, 150 mM NaCl). Protein concentrations were determined by the bicinchoninic acid (BCA) method, with bovine serum albumin as the standard. Enzyme fractions were analyzed by 12% sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) and visualized with Coomassie brilliant blue.38 Construction of GAD Truncation Mutants. GAD truncation mutants were generated by PCR using pET-28a-GAD as a template. Reverse primers containing the mutation were used: 5′-GAATTCTCATCCGTATTTCTTAGGTGC-3′ (for GAD Δ3) and 5′GAATTCTCAATFATGATAGACAATGTG-3′ (for GAD Δ11). The forward primers were the same as those used to clone the gadB gene originally (see above). PCR was performed as described above. The amplified plasmid was digested with NdeI and EcoRI and transformed into E. coli DH5α cells. Clones were sequenced to verify the presence of the mutation. The cloned plasmids were transformed into E. coli BL21(DE3) cells and expressed. The truncated mutants (Δ3 and Δ11) expressed in E. coli were purified and characterized as described above. Enzyme Activity Assay and Statistical Analysis. Enzyme activity was determined by measuring the amount of GABA produced by GAD through HPLC analysis. The reaction mixture (100 μL) contained 0.022 mM PLP, 15 mM glutamate, and an appropriate amount of GAD (≈10 μg) in 100 mM sodium acetate buffer (pH 5.0) or 100 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) buffer (pH 7.0). After a 20 min incubation at 37 °C, the enzyme reaction was stopped by the addition of ice-chilled 100% EtOH (600 μL) to the reaction mixture. After a centrifugation at 15000g for 5 min at 4 °C, the resulting supernatant (120 μL) was mixed with 380 μL of o-phthalaldehyde (OPA) solutions (pH 9.3) comprising 1.0 mL of methanolic OPA (20 mM OPA in 50% (v/v) methanol), 250 μL of borate buffer (the 1:1 (v/v) ratio of 0.2 M NaOH and 0.2 M boric acid dissolved in 0.2 M KCl, pH 9.9), and 25 μL of 2-mercaptoethanol. To detect the amine group, the reaction mixture was further incubated at room temperature for 8 min and then analyzed using the Waters Alliance HPLC system (model 2695; Milford, MA, USA) equipped with a Dual λ absorbance detector (model 2487; Waters) with a C18 column (SP̂ Column, 250 × 4.6 mm, 5 μm particle size). The mobile phase was a 20% (v/v) acetonitrile solution containing 20 mM sodium acetate and 0.02% (v/v) triethylamine. The column temperature was set at 25 °C and eluted at 0.8 mL/min. The reaction products (i.e., GABA) were monitored by measuring the A338 nm, and the amount of GABA was estimated from the integrated peak area based on the HPLC data. One unit of GAD activity is defined as the amount of enzyme that produces 1 μmol of product per minute under the assay conditions. All experiments were carried out in duplicate. For kinetic studies, the kinetic parameters of GAD were determined in 100 mM sodium acetate buffer (pH 5.0) or 100 mM PIPES buffer (pH 7.0), by varying the substrate concentration (1−300 mM) for 5 min at 40 °C. Kinetic data were analyzed using Michaelis−Menten equations by fitting data with the Origin 8.0 program. Biochemical Characterization of L. plantarum GAD. To determine the effect of temperature on the enzyme activity, the enzyme was incubated in 100 mM sodium acetate (pH 5.0) containing 15 mM glutamate for 20 min at a temperatures from 30 to 60 °C. For a

During the past decade, a variety of microbial GADs were expressed and characterized,34−37 and several crystal structures were determined to investigate the pH dependence of catalytic activity.12 Consequently, it was found that the N-termini and/or the C-termini of GAD might be responsible for the pHinduced conformational change for the regulation of catalytic activity in the acidic environment. Herein, we overexpressed the gadB gene encoding GAD from Lactobacillus plantarum in E. coli and characterized it. Subsequently, on the basis of the model structure and computational analysis of L. plantarum GAD, we attempted to generate C-terminally truncated mutant enzymes and investigated the pH dependence of enzyme activity, underlying the important role of C-terminal region for the GAD activity at low pH values.

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MATERIALS AND METHODS

Materials. Restriction enzymes, prime star DNA polymerase, deoxynucleotide triphosphates, and chemicals for PCR were obtained from Takara Biomedicals (Takara Co., Shiga, Japan). The pTOPV2 blunt vector for cloning was obtained from Enzynomics (Daejeon, Korea). The pET-28a expression vector and a His-bind resin kit were obtained from Novagen (Madison, WI, USA). Genomic-tip, gel extraction, PCR purification, and plasmid miniprep kit were obtained from Qiagen (Hilden, Germany). Electrophoresis reagents were obtained from Bio-Rad (Hercules, CA, USA). All chemicals used for enzyme assays and characterization were obtained from Sigma (St. Louis, MO, USA). Oligonucleotides were synthesized by Bioneer (Daejeon, Korea). Bacterial Strains and Culture Conditions. L. plantarum ATCC 14917 was obtained from American Type Culture Collection (ATCC). The strain was grown in De Man, Rogosa, and Sharpe (MRS) medium at 30 °C for 72 h. E. coli strain DH5α was used as a host for the construction of expression vector. E. coli BL21 (DE3) was used as an expression host for GAD from L. plantarum. Both E. coli strains were grown in Luria−Bertani (LB) medium with kanamycin (50 μg/mL) in a rotary shaker at 37 and 25 °C, respectively. Plasmid pTOPV2 blunt vector was used as a cloning and sequencing vector, and pET-28a was used for expression. Cloning and Expression of L. plantarum GAD. From a search of the microbial genome sequences in GenBank (accession no. CCC80401.1), we found a gadB-encoding gene in L. plantarum. Genomic DNA was isolated from L. plantarum and purified using a genomic DNA extraction kit (Qiagen) according to the manufacturer’s instructions. The gene-encoding GAD was amplified by PCR from L. plantarum genomic DNA using the forward/reverse primer pair 5′-CATATGGCAATGTTATACGGTAAACAC-3′ and 5′-GAATTCTCAGTGTGTGAATCCGTATTTC-3′. A NdeI site was incorporated into the forward primer and an EcoRI site into the reverse primer for cloning into pET-28a. PCR was performed in 10× prime star buffer containing 2 mM MgCl2, 20 ng of DNA, 10 pmol of each primer, 200 μM dNTP mix, and 2.5 U of prime star DNA polymerase in a total volume of 50 μL. After initial denaturation for 4 min at 94 °C, DNA was amplified over 30 cycles (30 s of denaturation at 94 °C, 30 s of annealing at 57 °C, and 1 min of extension at 72 °C), followed by a final extension step of 5 min at 72 °C. The PCR product was cloned into the pTOPV2-blunt vector and transformed into E. coli DH5α. Transformants containing the pTOPV2-blunt vector harboring the L. plantarum gadB gene were selected on LB medium−ampicillin plates. Plasmid DNA isolated from cells containing the gene was digested with NdeI and EcoRI. The released gadB gene was then ligated into the NdeI and EcoRI sites of the pET-28a vector, which directs the formation of fusion proteins with N-terminus polyhistidine (6× His) to generate pET-28a-GAD. For expression of the recombinant enzyme, E. coli BL21(DE3) cells transformed with pET-28a-GAD were grown in an LB medium (0.5 L) containing 50 μg of kanamycin per milliliter at 25 °C induced at midexponential phase (A600 ∼ 0.6) with 1 mM IPTG, grown for an additional 12 h, and harvested by centrifugation (10000g, 20 min, 4 °C). Bacterial pellets were stored at −70 °C until use. B

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thermostability test, the enzyme was incubated at various temperatures (40−90 °C) in the presence of 100 mM sodium acetate (pH 5.0) for different periods of time (0−9 h). The residual activity was measured under the same conditions as described above. The pH optimum of the enzyme was determined using the 100 mM sodium acetate buffer and 100 mM PIPES buffer at pH levels from 3.5 to 5.5 and from 5.5 to 6.0, respectively, at 37 °C. To determine pH stability, the enzyme solution was preincubated in each buffer at room temperature for different periods of time (0−96 h), and the residual activity was measured under same conditions as described above. All experiments were performed in triplicate. Sequence Alignment and Homology Modeling of GAD Proteins. In a search of the microbial genome sequences in NCBI, we obtained the amino acid sequence of L. plantarum GAD (NCBI protein database accession no. CCC80401.1). The sequence was used to find a suitable template for homology modeling from the Brookhaven Protein Data Bank (PDB) using the software BLAST, revealing that L. plantarum GAD had an amino acid sequence identity of 42.6% (similarity of 57.2%) to the crystal structure of E. coli GAD (PDB ID 1PMO).12 On the basis of the E. coli GAD structure as a template, homology modeling of L. plantarum GAD was performed using the program Modeler39 in the Homology module of Insight II (Accelrys Inc., San Diego, CA, USA) on a Silicon Graphics R10000 workstation. The dimeric model of L. plantarum GAD thus built was subjected to energy minimization by CG using the program DISCOVER (MSI/ Accelrys) with the consistent-valence force field (CVFF) until the structure reached the final derivative of 0.001 kcal/mol. To study the interaction between L. plantarum GAD and glutamate as the substrate, the crystal structure (PDB code 1PMO) of glutamate bound to GAD was used. The model of glutamate was subjected to energy refinement using the program DISCOVER with CVFF until the structure reached the final derivative of 0.001 kcal/mol. The model of L. plantarum GAD was then docked to the glutamate using the Patchdock server.40 The docked structures that yielded the best score were selected and analyzed visually using the software Insight II. Molecular dynamics (MD) simulations were performed on the docked structures to predict the favorable binding interactions. The docked complexes of L. plantarum GAD and glutamate were solvated by a 10 Å water layer by the program SOAK of Insight II. The resulting systems were subsequently energy minimized using Steepest Descent (SD) and DISCOVER. The minimized systems were equilibrated for a period of 210 ps with constant volume and temperature (NVT ensemble, at 298 K) through the velocity verlet integrator.41 The time step for integration was 1 fs using the RATTLE algorithm, a velocity version of SHAKE.42 The last 120 ps trajectories were analyzed, saving the coordinates at 0.2 ps intervals. All of the simulations were performed with CVFF force fields; for nonbonded calculations, the cell multipole method along with a dielectric constant of 1 was used. The stereochemical qualities of the final structures were analyzed with PROCHECK. To elucidate the interactions responsible for the dimerization of the protein as well as between the protein and its substrate (i.e., GAD and its substrate glutamate), the What If software package40 and the Biopolymer module of Insight II were used. Thin-Layer Chromatography Analysis. TLC analysis of monosodium glutamate (Glu) and GABA was performed in n-butanol/acetic acid/water (5:3:2 by volume) as a solvent for separation by the ascending technique on 0.2 mm, silica gel-coated, aluminum sheets (type 60; Merck, Deamstadt, Germany). The plate was sprayed with 60% (w/v) ninhydrin and then heated to visualize the spots.43

Figure 1. Purification of the recombinant L. plantarum GADs. Purified wild-type GAD and its truncated mutants were analyzed by SDSPAGE (left panel) and native-PAGE (right) (A) and by gel filtration chromatography (B). Lanes: M, molecular weight markers (reduced); 1, whole-cell proteins; 2, cytosolic fractions; 3, purified L. plantarum GAD; 4, purified L. plantarum GAD (Δ3); 5, purified L. plantarum GAD (Δ11); 6, purified L. plantarum GAD (nonreduced); M′, molecular weight markers (nonreduced). Gel filtration chromatography was performed using a Superdex 200 16/60 column, which was run at a flow rate of 1 mL/min, and the elution profile was monitored at 280 nm. The column was calibrated with blue dextran (2000 kDa), thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), and ovalbumin (44 kDa) as standards.

upon induction with IPTG (Figure 1A). A single Ni2+ chelate affinity chromatography step served to purify the expressed L. plantarum GAD protein to >95% purity. After removal of the His-tag from the fusion protein by treatment with thrombin, we further purified the intact GAD through gel filtration chromatography (Figure 1B). The apparent Mr of the fusion protein was estimated to be 53 kDa by SDS-PAGE, consistent with the Mr (53 749 Da) calculated from the presumptive amino acid sequence (Figure 1A). However, native-PAGE analysis and gel filtration chromatography on Superdex 200 demonstrated that the purified enzyme was estimated to be about 110 kDa, suggesting a homodimeric structure (Figure 1B). On the basis of the fact that both truncated mutant enzymes were also observed as homodimers, we concluded that truncated mutation did not affect the oligomeric state of both mutant GADs. Biochemical Characterization of L. plantarum GAD. The temperature and pH dependences of the recombinant GAD were determined by using HPLC after a 20 min incubation at various temperatures and pH values. Purified GAD had optimal activity at pH 5.0, and its optimum temperature for GAD activity was 40 °C (Figure 2). To investigate the effects of pH on the stability of L. plantarum GAD, the purified enzyme

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RESULTS AND DISCUSSION Cloning, Expression, and Purification of L. plantarum GAD. The gadB gene encoding GAD was amplified by PCR from L. plantarum ATCC 14917 genomic DNA and cloned into the pTOPV2 blunt cloning vector. For expression in E. coli BL21(DE3) as well as to facilitate subsequent purification, the gadB gene was cloned into the pET-28a expression vector, resulting in pET-28a-GAD. The cloned gene was successfully expressed as an N-terminal hexa-histidine-tagged fusion protein C

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Figure 2. Effects of pH (A) and temperature (B) on L. plantarum GAD activity. (A) L. plantarum GAD activity was measured in 100 mM sodium acetate buffer at pH 3.5−5.5 and 100 mM piperazineN,N′-bis(2-ethanesulfonic acid) (PIPES) buffer at pH 6.0−7.0, respectively. (B) L. plantarum GAD activity was determined at various temperatures (30−60 °C) in 50 mM sodium acetate buffer at pH 5.0. All experiments were performed in triplicate. Error bars indicate the standard deviation. Figure 3. Effects of pH (A) and temperature (B, C) on the stability of L. plantarum GAD. (A) The purified L. plantarum GAD enzyme was preincubated in buffers of various pH values at room temperature for different periods of time (0−96 h) prior to determination of enzyme activity. (B) The purified L. plantarum GAD enzyme was incubated at various temperatures (40−90 °C) in 100 mM sodium acetate (pH 5.0) for different periods of time (0−9 h) prior to assessment of the enzyme activity. (C) Irreversible thermal unfolding of L. plantarum GAD was monitored by circular dichroism (CD) ellipticity at 222 nm in a 1.0 cm light path quartz glass cuvette at various temperatures ranging from 25 to 110 °C at a heating rate of 1 °C/min. CD measurements were carried out in a Jasco J-810 spectropolarimeter with a Peltier temperature-controlled cuvette holder. Protein concentration was 1 mg/mL in 50 mM sodium acetate buffer. The ratio of denatured and total protein in the transition range, fd = (εN − ε)/(εN − εU), was calculated from their CD signal ε relative to the signal of the native and unfolded baseline εN and εU. Melting temperature (Tm) is defined as the midpoint transition of unfolding of GAD.

was preincubated at various pH values ranging from pH 3.5 to 6.0 for 96 h at 37 °C, and then their residual activities were measured (Figure 3A). For thermostability, purified enzymes were preincubated at various temperatures ranging from 40 to 90 °C, and the residual activity was also determined under the standard assay conditions (Figure 3B). As expected, incubation of L. plantarum GAD at pH values in the range of pH 4.0−5.5 caused no significant loss of GAD activity after a 96 h incubation (>90% of original activity). After a 9 h incubation of L. plantarum GAD at various temperatures in the range from 40 to 60 °C, the recombinant enzyme retained >95% of original activity, indicating that L. plantarum GAD is thermostable. Indeed, the recombinant enzyme with a thermal half-life of 46 min at 90 °C retained approximately 70% of initial activity even after incubation at 70 °C for 9 h, whereas the enzyme was rapidly inactivated at 100 °C. Therefore, these data indicated that the gadB gene from L. plantarum encodes a thermostable GAD, the structure of which is quite stable at acidic pH and elevated temperatures.

To further investigate the effect of temperature on the stability of L. plantarum GAD in more detail, we performed D

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Table 1. Biochemical Properties and Kinetic Parameters of L. plantarum and Other Microbial GADsa microorganism Lactobacillus plantarum Lactobacillus brevis IFO 12005 Lactobacillus brevis 877G Lactobacillus paracasei Lactococcus lactis Neurospora crassa Escherichia coli a

Topt (°C) 40 30 45 50 30

pHopt

Km (mM)

kcatb (min−1)

kcat /Km (mM−1 min−1)

ref

4.5 4.2 5.2 5 4.7 5.0 4.4

22.8 ± 1.1 9.3 3.6 5 0.5 2.2 1.0

1304.8 ± 23.3 390 17.7 428

57.2 ± 1.8 42 4.9 85.5

this study 19 34 36 45 46 44

a

Data are means ± standards deviations. bkcat is the number of substrate molecules reacted per active site per minute.

Figure 4. Model structure of the dimeric L. plantarum GAD and its detailed views. (A) The dimeric L. plantarum GAD is shown using cylindrical representations. The modeled structure of L. plantarum GAD was generated on the basis of the crystal structure of E. coli GAD (PDB code 1PMO) using the SWISS-MODEL program. One monomer (M1, left) is in pale yellow, whereas the other monomer (M2, right) is in pale pink. (B, C) Close-up view of the intersubunit contact regions between the two monomers. N-terminal (1−60 residues, E. coli numbering) and C-terminal (Pro452−Thr466, E. coli numbering) regions of the monomer (M2) of the L. plantarum GAD are in red, and the loop regions (Ile85−Tyr90 and Val300−Phe313, E. coli numbering) of the other monomer (M1) are in cyan. (D) The conformation of GAD at normal pH (i.e., pH 7.0) involves C-terminal residues of the protein (colored white) occupying the substrate binding site. Amino acid residues in L. plantarum GAD are in blue. (E) At low pH (i.e., pH 4.5), GAD undergoes a conformational change. Red dotted lines indicate hydrogen bonds. Amino acids with asterisks refer to residues specific to the L. plantarum GAD sequence. E

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thermal unfolding experiments using circular dichroism (CD). Thermal unfolding of enzyme was initiated at temperatures beyond the optimal temperature for growth of the corresponding bacterium. Consequently, L. plantarum GAD had the apparent melting temperature (Tm) of 95 °C (Figure 3C). Together with the thermostability data above, this unfolding experiment clearly demonstrated that L. plantarum GAD is not only acidophilic but also thermostable. To determine the kinetic parameters of L. plantarum GAD for glutamate as the substrate, we measured GAD activity after 5 min of incubation at 40 °C as described above. As a result, the Km for glutamate was 22.8 mM, and the Vmax was 24.4 U/mg. The catalytic efficiency (kcat/Km) for glutamate was 57.2 mM−1 min−1 (Table 1). As expected, L. plantarum GAD also showed very similar physicochemical properties (i.e., Topt and pHopt), in comparison with other microbial GADs including E. coli GAD.44 However, L. plantarum GAD had at least a kcat value that is about 3 times higher than for other GADs (Table 1). Homology Modeling and Truncation Mutagenesis of L. plantarum GAD. Comparative sequence analysis between E. coli and L. plantarum GADs was performed by FASTA. L. plantarum GAD showed sequence similarity of about 57% with E. coli GAD encoded by the gadB gene. To provide a structural basis for further investigations of the L. plantarum GAD, a homology-based structural modeling was performed as described above (Figure 4). Because the active forms of other microbial GADs derived from Lactobacillus strains as well as E. coli GadB are known as a dimer and a hexamer, respectively,12,36 we generated the dimeric model of the L. plantarum GAD by superimposing two monomeric models separately into the corresponding monomeric units of E. coli GAD and subsequently merging them together by the Biopolymer module of Insight II. To study the interaction between L. plantarum GAD and glutamate as the substrate, the crystal structure (PDB code 1PMO) of glutamate bound to GAD was used. Previously, it has been reported that E. coli GAD undergoes a pH-dependent conformational change and exhibits optimal activity at low pH.12 The GAD enzyme is localized exclusively to the cytoplasm at neutral pH but is recruited to the membrane when pH levels fall. Indeed, our structural modeling by the Insight II program suggested that the Ile85−Tyr90 (E. coli numbering) region in M1 adjacent to the other GAD subunit (M2) is where conformational changes occur by mainly Asp86 and Glu89 (E. coli numbering) residues in a pH-dependent manner (Figure 4). Intriguingly, such a pH-induced conformational change is accompanied by displacement of the loop region (Val300−Phe313 residues, E. coli numbering) away from the entrance site for the substrate at low pH values. Simultaneously, the C-terminal loop region (Pro452−Thr466, E. coli numbering) was also pushed away to open the entrance for the substrate binding site at low pH, resulting in the formation of active GAD (Figure 4B,C). On the other hand, both the 300− 313 region in M1 and the C-terminal Pro452−Thr466 loop region in M2 occupy the substrate binding site tunnel at neutral pH, resulting in the inactive GAD. These simulation data strongly suggested that the C-terminal loop region may play an important role in the regulation of GAD activity through pH-induced conformational changes. Therefore, we examined whether the C-terminal loop region is mainly involved in the regulation of GAD activity through pH-dependent conformational change. For this, we generated two C-terminally truncated mutants (i.e., GAD Δ3 and GAD Δ11), which contain deletions of 9 and 33 nucleotides, respectively. Notably,

Figure 5. pH dependence of catalytic activity for L. plantarum GAD and its truncated mutants. After incubation of the wild-type L. plantarum GAD and its C-terminally truncated mutants (Δ3 and Δ11) with monosodium glutamate (Glu) as a substrate at different pH values, reaction mixtures were analyzed by (A) TLC and (B) HPLC. (C) Comparison of relative enzyme activities for the wild-type and mutant GADs at pH 5 and 7. The specific activities for the wild-type (8.9 U/mg) and the mutant Δ11 (1.3 U/mg) were set at 100% at pH 5 and 7, respectively.

it was found that the expression level of both truncated mutants in E. coli at 25 °C was much higher than that at 37 °C (data not shown). Thus, we purified both mutant enzymes expressed in E. coli at 25 °C. After purification of those mutants, their enzymatic activities were then determined on the basis of the production of GABA in a pH range of 3.5−8.0 as judged by thin-layer chromatography. As shown in Figure 5A, the wildtype GAD showed its GABA conversion activity under acidic conditions, indicating that it became an active form at low pH F

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Table 2. Kinetic Parameters for the Wild-Type GAD and Its Truncated Mutantsa enzyme

pH

Vmax (U/mg)

Km (mM)

kcatb (min−1)

kcat /Km (mM−1 min−1)

WT

5.0 7.0

24.4 ± 0.4 ndc

22.8 ± 1.1 122.1 ± 3.8

1304.8 ± 23.3 0.91 ± 0.01

57.2 ± 1.8 nd

Δ11

5.0 7.0

45.1 ± 2.9 1.3 ± 0. 1

33.8 ± 4.3 137.1 ± 11.2

2415.2 ± 156.5 29.1 ± 0.9

72.1 ± 4.6 0.2 ± 0.1

Data are means ± standards deviations. The kinetic parameters were obtained by fitting the experimental data with the Michaelis−Menten equation as follows: v = Vmax[S]/Km + [S]. bkcat is the number of substrate molecules reacted per active site per minute. cnd, not determined. a

of 5.0 for enzymatic activity) very similar to those of other microbial GADs in terms of the pH dependence of enzyme activity. Its 3D structural model suggested that the C-terminal loop region may be critical for the pH dependence of enzyme activity at low pH. Such a pH-induced GAD activity through conformational changes such as the molecular switch can be a useful engineering target for altering the pHopt of GAD, which allows us to create GAD mutants that are resilient to pH fluctuations. Therefore, this feature might be beneficial for engineering GAD with the broad range of pH optimum for its catalytic activity in the immobilization of the enzyme for the production of GABA.

values (pH 3.5−5.0). However, the enzyme did not show any activity at pH 5.5−6.0. On the other hand, both GAD Δ3 and GAD Δ11 exhibited pronounced enzyme activity in the broader pH range of 3.5−8.0. On the basis of these qualitative results, we also performed a HPLC analysis to compare the GAD activities for the wild-type and mutant enzymes (Figure 5B). At pH 5, both truncated enzymes showed lower activities than the wild-type enzyme, as expected. However, although the wildtype enzyme exhibited little activity, both mutants had pronounced GABA peaks at pH 7. Indeed, the amounts of GABA produced by Δ11 and Δ3 mutants were much higher than that of the wild-type enzyme (Figure 5C). These data clearly indicated that deletion of the Ile454−Thr468 region (corresponding to Pro452−Thr466 in E. coli GAD) at the C-terminus of L. plantarum GAD caused loss of sensitivity to change in pH values. In addition, together with the prediction data, these observations strongly suggested that the C-terminal region might function as a lid for the entrance of glutamate as the substrate, thereby regulating the GAD activity. Comparison of the pH Dependence of GAD Activity with Its Truncated Mutants. To further investigate the pH dependence of the C-terminal domain for GAD activity, we determined the kinetic parameters for the wild-type GAD and its Δ11 mutant at pH 5 and 7. As described in Table 2, the wild-type enzyme showed the Vmax value of 24.4 at pH 5, whereas its enzyme activity was negligible at pH 7 as expected and described elsewhere.22 GAD Δ11 showed higher activity than the wild-type at pH 5 (Figure 5C), which is clearly supported by the kinetic values (Table 2). This might be ascribed to the deletion of the C-terminal loop region, where glutamate is more freely accessible to the substrate binding site of GAD. Indeed, even at pH 7 at which there is no activity detected from the wild-type enzyme, the GAD Δ11 mutant showed a prominent level of enzyme activity (0.54 U/mg) with an unusually high kcat value (Table 2), although it had lower levels of activity than those of the wild-type and mutant enzymes at pH 5. To date, it has been noted that the critical feature of microbial GADs is the conformational change of a calmodulin (CaM)-binding domain in the C-terminal region of Saccharomyces cerevisiae and the N-termini of E. coli GadB for the acidic pH optimum.12,37 Therefore, our HPLC analysis and kinetic data clearly demonstrated that the C-terminus of L. plantarum GAD plays an important role in the regulation of enzyme activity for the pH alteration of environments. These data also provide biochemical evidence for the involvement of protein in functioning as a molecular switch for acid resistance as described in refs 12, 29, 37, and 44. In this study, we expressed the gadB gene-encoding GAD from L. plantarum as the generally recognized as safe (GRAS) strain and characterized the recombinant enzyme. Characterization of its physicochemical properties (i.e., the pHopt, tempopt, thermostability, Vmax, kcat, etc.) demonstrated that this L. plantarum GAD has properties (i.e., an acidic pH optimum

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AUTHOR INFORMATION

Corresponding Author

*(D.-W. L.) Phone: +82-53-950-5718. Fax: +82-53-953-7233. E-mail: [email protected]. Author Contributions ∥

S.-M.S. and H.K. contributed equally to the work.

Funding

This work was supported by the National Agenda Program for Agricultural R&D (PJ0097672014) from the Rural Development Administration and the Basic Science Research Program through the National Research Foundation of Korea (NRF2012R1A1A2042114), South Korea. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We greatly appreciate Do-Yoon Kim’s critical contribution to 3D modeling and structural analysis. REFERENCES

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