Recombinant Glutamate Decarboxylase Research Articles

An In Situ Assembled Trapping Gel Repairs Spinal Cord Injury by Capturing Glutamate and Free Calcium Ions.

Spinal cord injury (SCI) can lead to devastating autonomic dysfunction. One of the most challenging issues for functional repair in SCI is the secondary damage caused by the increased release of glutamate and free Ca2+ from injured cells. Here, an in situ assembled trapping gel (PF-SA-GAD) is developed to sweep glutamate and Ca2+ , promoting SCI repair. The hydrogel solution is a mixture of recombinant glutamate decarboxylase 67 (rGAD67) protein, sodium alginate (SA), and pluronic F-127 (PF-127). After intrathecal administration, temperature-sensitive PF-127 promoted in situ gelation. Glutamate (Glu) is captured and decarboxylated by rGAD67 into γ-aminobutyric acid (GABA). SA reacted with the free Ca2+ to generate gellable calcium alginate. Thereby, this in situ trapping gel retarded secondary neuron injury caused by Glu and free Ca2+ during SCI. In rat models of SCI, PF-SA-GAD reduces the lesion volume and inflammatory response after SCI, restores the motor function of rats with SCI. Together, the in situ assembled trapping gel is a long-term effective and minimally invasive sweeper for the direct elimination of glutamate and Ca2+ from injury lesions and can be a novel strategy for SCI repair by preventing secondary injury.

Expression, purification, and characterization of glutamate decarboxylase from human gut-originated Lactococcus garvieae MJF010.

Human gut-originated lactic acid bacteria were cultivated, and high γ-aminobutyric acid (GABA)-producing Lactococcus garvieae MJF010 was identified. To date, despite the importance of GABA, no studies have investigated GABA-producing Lactococcus species, except for Lc. lactis. A recombinant glutamate decarboxylase of the strain MJF010 (rLgGad) was successfully expressed in Escherichia coli BL21(DE3) with a size of 53.9kDa. rLgGad could produce GABA, which was verified using the silylation-derivative fragment ions of GABA. The purified rLgGad showed the highest GABA-producing activity at 35°C and pH 5. rLgGad showed a melting temperature of 43.84°C. At 30°C, more than 80% of the activity was maintained even after 7h; however, it rapidly decreased at 50°C. The kinetic parameters, Km, Vmax, and kcat, of rLgGad were 2.94 mM, 0.023 mM/min, and 12.3min- 1, respectively. The metal reagents of CaCl2, MgCl2, and ZnCl2 significantly had positive effects on rLgGad activity. However, most coenzymes including pyridoxal 5'-phosphate showed no significant effects on enzyme activity. In conclusion, this is the first report of Gad from Lc. garvieae species and provides important enzymatic information related to GABA biosynthesis in the Lactococcus genus.

Characterization of a novel glutamate decarboxylase (GAD) from Latilactobacillus curvatus K285 isolated from Gat -Kimchi.

A lactic acid bacteria (LAB) producing γ-aminobutyric acid (GABA) was isolated from Gat-Kimchi, a Korean vegetable food. The isolate, K285, was identified as Latilactobacillus (formly Lactobacillus) curvatus. The gadB encoding glutamate decarboxylase (GAD) was cloned and an ORF encoding a protein of 451 amino acids was located. K285 GAD was smaller than other LAB GADs, and its amino acid sequence showed less than 80% homology with other LAB GADs, indicating the uniqueness of K285 GAD. The gadC encoding glutamate/GABA antiporter was located 75bp upstream of gadB, indicating gadCB operon structure. The gadB was overexpressed in Escherichia coli and recombinant GAD was purified. Optimum pH and temperature of recombinant K285 GAD were pH 5.0 and 50°C, respectively, and the activity was dependent on pyridoxal 5'-phosphate. The Km and Vmax of GAD were 5.35 ± 0.27mM and 0.041 ± 0.0008mM/min, respectively. Lb. curvatus K285 might be useful for the production of foods enriched with GABA.

Characterization of three glutamate decarboxylases from Bacillus spp. for efficient \u03b3-aminobutyric acid production

BackgroundGamma-aminobutyric acid (GABA) is an important bio-product used in pharmaceuticals and functional foods and as a precursor of the biodegradable plastic polyamide 4. Glutamate decarboxylase (GAD) converts l-glutamate (l-Glu) into GABA via decarboxylation. Compared with other methods, develop a bioconversion platform to produce GABA is of considerable interest for industrial use.ResultsThree GAD genes were identified from three Bacillus strains and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction temperature and pH values for three enzymes were 40 °C and 5.0, respectively. Of the GADs, GADZ11 had the highest catalytic efficiency towards l-Glu (2.19 mM− 1 s− 1). The engineered E. coli strain that expressed GADZ11 was used as a whole-cell biocatalyst for the production of GABA. After repeated use 14 times, the cells produced GABA with an average molar conversion rate of 98.6% within 14 h.ConclusionsThree recombinant GADs from Bacillus strains have been conducted functional identification. The engineered E. coli strain heterologous expressing GADZ1, GADZ11, and GADZ20 could accomplish the biosynthesis of l-Glu to GABA in a buffer-free reaction at a high l-Glu concentration. The novel engineered E. coli strain has the potential to be a cost-effective biotransformation platform for the industrial production of GABA.

Molecular cloning, gene overexpression and characterization of glutamate decarboxylase from Enterococcus faecium DO

In most bacteria, Glutamate decarboxylase (GAD) activity significantly reduces at pH > 6. Since l-glutamate is neutral to slightly alkaline, a large amount of hydrochloric acid must be added to the reaction mixture. Acidic reaction conditions greatly increase the corrosion of production equipment and shorten the life of manufacturing equipment. Therefore, this study aims at studying the overexpression of recombinant GAD that is active at the neutral pH and high-level production of γ-aminobutyric acid. A gene sequence encoding GAD of Enterococcus faecium DO was cloned into PET21a (+) and was overexpressed in Escherichia coli BL21 (DE3). The construct was confirmed by colony PCR and double digested with BamHI and XhoI. The result from Western blot analysis showed a signal band at the molecular mass of 54 kDa. The specific activity of the recombinant GAD was 78.17 U mg−1 at 45 °C and pH = 6.6. It was reduced rapidly when pH > 7.8. The Km, Vmax, and kcat values of recombinant GAD were 1.8 mM, 12.9 μmol L−1 min−1, and 0.43 s−1, respectively. Therefore, recombinant GAD is a potent candidate for GABA production in food and pharmaceutical industries, because it increases the efficiency of GABA synthesis, especially in foods with a neutral pH range.

Characterization of the Recombinant Glutamate Decarboxylase of Lactobacillus brevis G144 Isolated from Galchi Jeotgal, a Korean Salted and Fermented Seafood

A γ-aminobutyric acid (GABA)-producing microorganism was isolated from galchi (hairtail fish, Trichiurus lepturus) jeotgal, a Korean salted and fermented seafood. The G144 isolate produced GABA excessively when incubated in MRS broth containing monosodium glutamate (MSG, 3%, w/v). G144 was identified as Lactobacillus brevis through 16S rRNA and recA gene sequencing. gadB and gadC encoding glutamate decarboxylase (GAD) and glutamate/GABA antiporter, respectively, were cloned and gadB was located downstream of gadC. The operon structure of gadCB was confirmed by reverse transcription (RT)-polymerase chain reaction. gadB was overexpressed in Escherichia coli and recombinant GAD was purified and its size was 54.4 kDa as evidenced by SDS-PAGE results. Maximum GAD activity was observed at pH 5.0 and 40℃ and the activity was dependent on pyridoxal 5''-phophate. The Km and Vmax of GAD were 8.6 mM and 0.01 mM/min, respectively.

Microbial Production and Enzymatic Biosynthesis of γ-Aminobutyric Acid (GABA) Using Lactobacillus plantarum FNCC 260 Isolated from Indonesian Fermented Foods

In the present study, we isolated and screened thirty strains of GABA (γ-aminobutyric acid)-producing lactic acid bacteria (LAB) from traditional Indonesian fermented foods. Two strains were able to convert monosodium glutamate (MSG) to GABA after 24 h of cultivation at 37 °C based on thin layer chromatography (TLC) screening. Proteomic identification and 16S rDNA sequencing using MALDI-TOF MS identified the strain as Lactobacillus plantarum designated as L. plantarum FNCC 260 and FNCC 343. The highest yield of GABA production obtained from the fermentation of L. plantarum FNCC 260 was 809.2 mg/L of culture medium after 60 h of cultivation. The supplementation of 0.6 mM pyridoxal 5’-phosphate (PLP) and 0.1 mM pyridoxine led to the increase in GABA production to 945.3 mg/L and 969.5 mg/L, respectively. The highest GABA production of 1226.5 mg/L of the culture medium was obtained with 100 mM initial concentration of MSG added in the cultivation medium. The open reading frame (ORF) of 1410 bp of the gadB gene from L. plantarum FNCC 260 encodes 469 amino acids with a calculated molecular mass of 53.57 kDa. The production of GABA via enzymatic conversion of monosodium glutamate (MSG) using purified recombinant glutamate decarboxylase (GAD) from L. plantarum FNCC 260 expressed in Escherichia coli was found to be more efficient (5-fold higher within 6 h) than the production obtained from fermentation. L. plantarum FNCC 260 could be of interest for the synthesis of GABA.

Isolation of γ-Aminobutyric Acid Producing <i>Lactobacillus brevis</i> T118 from <i>Sun-Tae Jeotgal</i> and Its Glutamate Decarboxylase Gene Cloning

A γ-aminobutyric acid (GABA) producing microorganism was isolated from Sun-Tae Jeotgal, a Korean traditional fermented seafood. Two thousand presumptive lactic acid bacteria (LAB) isolates were screened for GABA production by thin layer chromatography. One isolate, T118, produced GABA profusely, and identified as Lactobacillus brevis. Growth of Lb. brevis T118 was examined during 120 h cultivation in MRS broth under different conditions. Lb. brevis T118 grew well at 30-37℃, initial pH of 4-7, and up to 5% NaCl (w/v). A gene, gadB, encoding glutamate decarboxylase (GAD) was cloned by PCR. gadC encoding a glutamate/GABA antiporter was cloned and gadC located immediately upstream of gadB, indicating gadCB operon structure. The operon structure was confirmed by reverse transcription (RT)-PCR. gadB was overexpressed in Escherichia coli BL21 (DE3) and recombinant GAD was purified. The size of recombinant GAD was 54.4 kDa by SDS-PAGE, which matched well with the calculated size from the nucleotide sequence.

An In Vivo Targeted Deletion of the Calmodulin-Binding Domain from Rice Glutamate Decarboxylase 3 (OsGAD3) Increases \u03b3-Aminobutyric Acid Content in Grains

BackgroundGamma-aminobutyric acid (GABA) is a non-protein amino acid present in all living things. GABA is mainly synthesized from glutamate by glutamate decarboxylase (GAD). In plants the enzymatic activity of GAD is activated by Ca2+/calmodulin binding (CaMBD) at the C-terminus in response to various stresses, allowing rapid GABA accumulation in cells. GABA plays a central role in not only stress responses but also many aspects of plant growth and development as a signaling molecules. Furthermore, it is known to be a health-promoting functional substance that exerts improvements in life-style related diseases such as hypertension, diabetes, hyperlipidemia, and so on. Previous reports indicated that CaMBD found plant GADs possess an autoinhibitory function because truncation of GAD resulted in extreme GABA accumulation in plant cells. Therefore, we attempted a genetic modification of rice GAD via genome editing technology to increase GABA levels in the edible part of rice.ResultsIn this study, we focused on GAD3, one of five GAD genes present in the rice genome, because GAD3 is the predominantly expressed in seeds, as reported previously. We confirmed that GAD3 has an authentic Ca2+/CaMBD that functions as an autoinhibitory domain. CRISPR/Cas9-mediated genome editing was performed to trim the coding region of CaMBD off from the OsGAD3 gene, then introducing this transgene into rice scutellum-derived calli using an all-in-one vector harboring guide RNAs and CRISPR/Cas9 via Agrobacterium to regenerate rice plants. Out of 24 transformed rice (T1), a genome-edited rice line (#8_8) derived from two independent cleavages and ligations in the N-terminal position encoding OsGAD3-CaMBD and 40 bp downstream of the termination codon, respectively, displayed a AKNQDAAD peptide in the C-terminal region of the putative OsGAD3 in place of its intact CaMBD (bold indicates the trace of the N-terminal dipeptides of the authentic CaMBD). A very similar rice line (#8_1) carrying AKNRSSRRSGR in OsGAD3 was obtained from one base pair deletion in the N-terminal coding region of the CaMBD. Free amino acid analysis of the seeds (T2) indicated that the former line contained seven-fold higher levels of GABA than wild-type, whereas the latter line had similar levels to the wild-type, although in vitro enzyme activities of recombinant GAD proteins based on the GAD3 amino acid sequence elucidated from these two lines in the absence of Ca2+/bovine CaM were both higher than wild-type counterpart. In addition to high level of GABA in #8_8, the average seed weight per grain and protein content were superior to wild-type and #8_1.ConclusionsWe have successfully established GABA-fortified rice by using CRISPR/Cas9 genome editing technology. Modified rice contained seven-fold higher GABA content and furthermore displayed significantly higher grain weight and protein content than wild-type brown rice. This is the first report of the production of GABA-enriched rice via a genome editing.

In Silico Study of Different Signal Peptides to Express Recombinant Glutamate Decarboxylase in the Outer Membrane of Escherichia coli

Gamma amino butyric acid (GABA) is used as drugs, food ingredients, and dietary supplements. l-glutamate is converted to GABA by the decarboxylation reaction, which is catalyzed by the glutamate decarboxylase (GAD). Escherichia coli is widely being used to express proteins. However, without appropriate signal peptide, it cannot be applied for secretory proteins. Selecting a suitable signal peptide (SP) is a critical step in the secretory production of different proteins. In silico identification of suitable SP is a reliable and cost-effective alternative to experimental approaches. In previous studies, the localization of proteins was not considered and the SPs of periplasmic, membranes and extracellular were compared. Therefore, this study aimed to predict the best SP for the expression of recombinant GAD in the outer membrane of E. coli only. Also, we compared twelve servers to evaluate protein localization, solubility, and secretory pathway. In the present study, 127 SPs were taken from the Signal Peptide database. The localization site, physico-chemical properties, location of cleavage sites, regions and D-score of them were determined by ProtComp, ProtParam, and SignalP 3.0 and 4.1 servers, respectively. To rank SPs based on the secretion properties, PRED-TAT and SignalP 5.0 webservers were used. Based on the results, the localization site of 13 SPs was in the outer membrane of E. coli. Among them, the most suitable candidates seemed to be torT with a reasonably high D-score, aliphatic index, and GRAVY, followed by ccmH and then pspE. TorT accelerates GAD scale-up production and might be useful in future experimental research.

Characterization of a Glutamate Decarboxylase (GAD) from Enterococcus avium M5 Isolated from Jeotgal, a Korean Fermented Seafood.

To develop starters for the production of functional foods or materials, lactic acid bacteria producing γ-aminobutyric acid (GABA) were screened from jeotgals, Korean fermented seafoods. One isolate producing a high amount of GABA from monosodium L-glutamate (MSG) was identified as Enterococcus avium by 16S rRNA gene sequencing. E. avium M5 produced 18.47 ± 1.26 mg/ml GABA when incubated for 48 h at 37°C in MRS broth with MSG (3% (w/v)). A gadB gene encoding glutamate decarboxylase (GAD) was cloned and overexpressed in E. coli BL21 (DE3) using the pET26b (+) expression vector. Recombinant GAD was purified through a Ni-NTA column and the size was estimated to be 53 kDa by SDS-PAGE. Maximum GAD activity was observed at pH 4.5 and 55°C and the activity was dependent on pyridoxal 5'-phosphate. The Km and Vmax values of GAD were 3.26 ± 0.21 mM and 0.0120 ± 0.0001 mM/min, respectively, when MSG was used as a substrate. Enterococcus avium M5 secretes a lot of GABA when grown on MRS with MSG, and the strain is useful for the production of fermented foods containing a high amount of GABA.

Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid

Glutamate decarboxylase (GAD) catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA). In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP)-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V) activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C). Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C), superior thermostability (2.8-fold greater than that of GAD-C), and higher kcat/Km (1.6-fold higher than that of GAD-C). Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG) to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA.

Characterization of Glutamate Decarboxylase (GAD) from Lactobacillus sakei A156 Isolated from Jeot-gal.

A gamma-aminobutyric acid (GABA)-producing microorganism was isolated from jeot-gal (anchovy), a Korean fermented seafood. The isolate, A156, produced GABA profusely when incubated in MRS broth with monosodium glutamate (3% (w/v)) at 37°C for 48 h. A156 was identified as Lactobacillus sakei by 16S rRNA gene sequencing. The GABA conversion yield was 86% as determined by GABase enzyme assay. The gadB gene encoding glutamate decarboxylase (GAD) was cloned by PCR. gadC encoding a glutamate/GABA antiporter was located immediately upstream of gadB. The operon structure of gadCB was confirmed by RT-PCR. gadB was overexpressed in Escherichia coli BL21(DE3) and recombinant GAD was purified. The purified GAD was 54.4 kDa in size by SDS-PAGE. Maximum GAD activity was observed at pH 5.0 and 55°C and the activity was dependent on pyridoxal 5'-phosphate. The Km and Vmax of GAD were 0.045 mM and 0.011 mM/min, respectively, when glutamate was used as the substrate.

Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj-Apis362 for high gamma-aminobutyric acid production.

Gamma-aminobutyric acid (GABA) is an important bioactive compound biosynthesized by microorganisms through decarboxylation of glutamate by glutamate decarboxylase (GAD). In this study, a full-length GAD gene was obtained by cloning the template deoxyribonucleic acid to pTZ57R/T vector. The open reading frame of the GAD gene showed the cloned gene was composed of 1410 nucleotides and encoded a 469 amino acids protein. To improve the GABA-production, the GAD gene was cloned into pMG36e-LbGAD, and then expressed in Lactobacillus plantarum Taj-Apis362 cells. The overexpression was confirmed by SDS-PAGE and GAD activity, showing a 53 KDa protein with the enzyme activity increased by sevenfold compared with the original GAD activity. The optimal fermentation conditions for GABA production established using response surface methodology were at glutamic acid concentration of 497.973 mM, temperature 36°C, pH 5.31 and time 60 h. Under the conditions, maximum GABA concentration obtained (11.09 mM) was comparable with the predicted value by the model at 11.23 mM. To our knowledge, this is the first report of successful cloning (clone-back) and overexpression of the LbGAD gene from L. plantarum to L. plantarum cells. The recombinant Lactobacillus could be used as a starter culture for direct incorporation into a food system during fermentation for production of GABA-rich products.

Characterization of a glutamate decarboxylase (GAD) gene from Lactobacillus zymae.

Lactic acid bacteria (LAB) were isolated from Kimchi, a Korean traditional fermented vegetable food. LAB accumulating GABA (γ-aminobutyric acid) in the culture media were screened by TLC analysis. One isolate, GU240, produced the highest amount of GABA among the 3,000 isolates and identified as a Lactobacillus zymae strain. Glutamate decarboxylase (GAD) gene was cloned and over-expressed in E. coli BL21(DE3) using pET26b(+). The recombinant GAD was purified by using a Ni-NTA column. Its size was 53kDa by SDS-PAGE. Maximum GAD activity was at pH 4.5 and 41°C and the activity was dependent on pyridoxal 5'-phosphate. Km and Vmax of LzGAD were 1.7mM and 0.01mM/min, respectively, when glutamate was used as a substrate.

Overexpression and characterization of recombinant glutamate decarboxylase from Thermococcus kodakaraensis KOD1

Glutamate decarboxylase (GAD) (EC 4.1.1.15) catalyzes decarboxylation of glutamic acid to produce gammaaminobutyric acid (GABA). A putative gad gene (tk1814) from an archaeon Thermococcus kodakaraensis KOD1 was cloned and transformed into Escherichia coli to produce a bulk amount of recombinant GAD. Activity of the purified GAD was optimal at 90°C and pH 8.0. Optimal concentration of substrate for conversion into gamma-aminobutyric acid by recombinant GAD was 50 mM monosodium glutamate. Recombinant GAD was confirmed to be monomeric, and its activity was greatly inhibited by various salts such as sodium chloride, Tris-HCl, and sodium phosphate. Km, Vmax, and Kcat values were 9.92 mM, 153.8 μmol min−1 mg−1, and 6.613×103 min−1 respectively.

Enhanced γ-aminobutyric acid-forming activity of recombinant glutamate decarboxylase (gadA) from Escherichia coli

γ-aminobutyric acid (GABA) is an important bioactive regulator, and its biosynthesis is primarily through the α-decarboxylation of glutamate by glutamate decarboxylase (GAD). The procedures to obtain GABA by bioconvertion with high activity recombinant Escherichia coli GAD have been seldom understood. In this study, Escherichia coli GAD (gadA) was highly expressed (about 70–75% of total protein) as soluble protein in Escherichia coli BL21(DE3) containing pET28a-gadA, which was induced by 0.4 mM IPTG in LB medium, and maximal GABA-forming activity of the recombinant GAD was 40 U/mL at a concentration (0.15 mM) of pyridoxal phosphate (PLP) and a concentration (0.6 mM) of Ca2+ at optimal pH of 3.8. The optimal concentration (7.5 mM) of Mn2+ can also improve the activity of recombinant enzyme, but the co-effect of Ca2+ and Mn2+ exhibited antagonism effect when added simultaneously. LB and 0.1% (w/v) lactose were selected as culture medium and inducer, respectively. The relative activity was markedly higher activated by Ca2+ (174%), Mn2+ (164%) than that by other seven bivalent cations. Finally, the yield of GABA was high of 94 g/L detected by paper chromatography or HPLC in 1 L reaction system with 30 mL crude GAD (12 U/mL). By entrapping Escherichia coli glutamate decarboxylase into sodium alginate and carrageenan gel beads, the activity of immobilized GAD (IGAD) remained 85% during the initial five batches and the activity still remained 50% at the tenth batch, these results indicated that the recombinant Escherichia coli GAD was feasible for the future industrial production of GABA.

Glutamate Decarboxylase fromLactobacillus brevis: Activation by Ammonium Sulfate

In this study, the glutamate decarboxylase (GAD) gene from Lactobacillus brevis IFO12005 (Biosci. Biotechnol. Biochem., 61, 1168-1171 (1997)), was cloned and expressed. The deduced amino acid sequence showed 99.6% and 53.1% identity with GAD of L. brevis ATCC367 and L. lactis respectively. The His-tagged recombinant GAD showed an optimum pH of 4.5-5.0, and 54 kDa on SDS-PAGE. The GAD activity and stability was significantly dependent on the ammonium sulfate concentration, as observed in authentic GAD. Gel filtration showed that the inactive form of the GAD was a dimer. In contrast, the ammonium sulfate-activated form was a tetramer. CD spectral analyses at pH 5.5 revealed that the structures of the tetramer and the dimer were similar. Treatment of the GAD with high concentrations of ammonium sulfate and subsequent dilution with sodium glutamate was essential for tetramer formation and its activation. Thus the biochemical properties of the GAD from L. brevis IFO12005 were significantly different from those from other sources.

C-terminal residues of plant glutamate decarboxylase are required for oligomerization of a high-molecular weight complex and for activation by calcium/calmodulin

Bacterial glutamate decarboxylase (GAD) is a homohexameric enzyme of about 330 kDa. Plant GAD differs from the bacterial enzyme in having a C-terminal extension of 33 amino acids within which resides a calmodulin (CaM)-binding domain. In order to assess the role of the C-terminal extension in the formation of GAD complexes and in activation by Ca 2+/CaM, we examined complexes formed with the purified full-length recombinant petunia GAD expressed in E. coli, and with a 9 amino acid C-terminal deletion mutant (GADΔC9). Size exclusion chromatography revealed that the full-length GAD formed complexes of about 580 kDa and 300 kDa in the absence of Ca 2+/CaM, whereas in the presence of Ca 2+/CaM all complexes shifted to ∼680 kDa. With deletion of 9 amino acids from the C-terminus (KKKKTNRVC 500), the ability to bind CaM in the presence of Ca 2+, and to purify it by CaM-affinity chromatography was retained, but the formation of GAD complexes larger than 340 kDa and enzyme activation by Ca 2+/CaM were completely abolished. Hence, responsiveness to Ca 2+/CaM is associated with the formation of protein complexes of 680 kDa, and requires some or all of the nine C-terminal amino acid residues. We suggest that evolution of plant GAD from a bacterial ancestral enzyme involved the formation of higher molecular weight complexes required for activation by Ca 2+/CaM.

Structure of Glutamate Decarboxylase from E. coli: Spectral Studies

Structure of recombinant glutamate decarboxylase (GADa) was studied by optical methods and electron microscopy. The active (pH 4.6) and inert (pH 6.3) holoGAD and apoGAD were investigated. Absorption and CD spectra were recorded in the range of 190—500 nm. Visible spectra were resolved into the bands corresponding to individual electron transitions using lognormal curves. The structures of predominant tautomers of internal aldimines were determined as ketoenamine at pH 4.6 and enolimine at pH 6.3. CD spectra show that holoGAD and apoGAD exhibit a negative band at 204—245 nm and a positive band near 190—204 nm. The contents of the secondary structure elements were estimated on the basis of the values of the mean residue ellipticity. Evidently, the main difference between the GAD forms studied is in the content of a-helix and random coil. HoloGAD has 50% of α- helix at pH 4.6 and 67% at pH 6.3, whereas apoGAD—17 and 27%, respectively. Thus presented data establish the essential role of pyridoxal phosphate (PLP) in the organization of the GAD secondary structure due to tightening its polypeptide chain. It seems possible, that conformational changes induced by PLP binding stabilize the protein structure and promote the assembly of subunits into macromolecule, which was confirmed by electron microscopy.