Preparation of magnetic polyacrylamide hydrogel with chitosan for immobilization of glutamate decarboxylase to produce γ-aminobutyric acid.

Gamma-aminobutyric acid (GABA) is an vital neurotransmitter, and the reaction to obtain GABA through biocatalysis requires coenzymes, which are therefore limited in the production of GABA. In this study, polyacrylamide hydrogels doped with chitosan and waste toner were synthesized for glutamate decarboxylase (GAD) and coenzyme co-immobilization to realize the production of GABA and the recovery of coenzymes. Enzymatic properties of immobilized GAD were discussed. The immobilized enzymes have significantly improved pH and temperature tolerance compared to free enzymes. In terms of reusability, after 10 repeated reuses of the immobilized GAD, the residual enzyme activity of immobilized GAD still retains 100% of the initial enzyme activity, and the immobilized coenzyme can also be kept at about 32%, with better stability and reusability. And under the control of no exogenous pH, immobilized GAD showed good performance in producing GABA. Therefore, in many ways, the new composite hydrogel provides another way for the utilization of waste toner and promises the possibility of industrial production of GABA.

Autoantibodies directed against glutamate decarboxylase interfere with glucose-stimulated insulin secretion in dispersed rat islets.

Islet autoantibodies, including autoantibodies directed against the 65kDa isoform of glutamate decarboxylase (GAD65Ab), are present in the majority of patients with newly diagnosed type 1 diabetes (T1D). Whereas these autoantibodies are historically viewed as an epiphenomenon of the autoimmune response with no significant pathogenic function, we consider in this study the possibility that they impact the major islet function, namely glucose-stimulated insulin secretion. Two human monoclonal GAD65Ab (GAD65 mAb) (b78 and b96.11) were investigated for uptake by live rat beta cells, subcellular localization and their effect on glucose-stimulated insulin secretion. The GAD65 mAbs were internalized by live pancreatic beta cells, where they localized to subcellular structures in an epitope-specific manner. Importantly, GAD65 mAb b78 inhibited, while GAD65 mAb b96.11 enhanced, glucose-stimulated insulin secretion (GSIS). These opposite effects on GSIS rule out non-specific effects of the antibodies and suggest that internalization of the antibody leads to epitope-specific interaction with intracellular machinery regulating insulin granule release. The most likely explanation for the alteration of GSIS by GAD65 Abs is via changes in GABA release due to inhibition or change in GAD65 enzyme activity. This is the first report indicating an active role of GAD65Ab in the pathogenesis of T1D.

Characterization and mutagenesis of a novel Mycobacterium smegmatis-derived glutamate decarboxylase active at neutral pH.

γ-aminobutyric acid (GABA) has various physiological functions and is widely used in medicine, food, and other fields. Glutamate decarboxylase (GAD) is a key enzyme that catalyzes the decarboxylation of L-glutamate to synthesize GABA. However, the industrial application of microorganism-derived GAD is limited by its rapid loss of enzymatic activity with pH approaching neutrality. In this study, a novel glutamate decarboxylase, GADMSM, from Mycobacterium smegmatis was overexpressed and purified. On the basis of homologous modeling and substrate molecular docking, several GADMSM mutants were constructed, and their enzymatic properties were analyzed. The results showed that the optimal pH of wild-type GADMSM is 5.4; at pH 6.2, 22.8% enzymatic activity was retained. The T211I replacement in GAD and C-terminal deletion mutant GADMSMΔC showed relatively high catalytic activity in a pH range of 5.0-7.0. The Vmax and Km values of GADMSMΔC were 14.69 and 5.70, respectively, at pH 5.5, and 9.87 and 6.17, respectively, at pH 7.0. Compared with the wild-type GAD, GADMSMΔC maintained higher affinity and enzymatic activity of the substrate, maintaining 78.5% of the highest enzymatic activity even at pH 7.0, which is the highest reported activity retention for GAD under neutral pH condition. Therefore, GADMSMΔC can be used for the transformation of high-yielding strains and industrial production of GABA.

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 53.9 kDa. 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 7 h; 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 12.3 min- 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.

Constitutive inorganic pyrophosphatase as a reciprocal regulator of three inducible enzymes in Escherichia coli.

BACKGROUND: Previous studies have demonstrated the formation of stable complexes between inorganic pyrophosphatase (PPase) and three other Escherichia coli enzymes - cupin-type phosphoglucose isomerase (cPGI), class I fructose-1,6-bisphosphate aldolase (FbaB) and l-glutamate decarboxylase (GadA). METHODS: Here, we determined by activity measurements how complex formation between these enzymes affects their activities and oligomeric structure. RESULTS: cPGI activity was modulated by all partner proteins, but none was reciprocally affected by cPGI. PPase activity was down-regulated upon complex formation, whereas all other enzymes were up-regulated. For cPGI, the activation was partially counteracted by a shift in dimer ⇆ hexamer equilibrium to inactive hexamer. Complex stoichiometry appeared to be 1:1 in most cases, but FbaB formed both 1:1 and 1:2 complexes with both GadA and PPase, FbaB activation was only observed in the 1:2 complexes. FbaB and GadA induced functional asymmetry (negative kinetic cooperativity) in hexameric PPase, presumably by favoring partial dissociation to trimers. CONCLUSIONS: These four enzymes form all six possible binary complexes in vitro, resulting in modulated activity of at least one of the constituent enzymes. In five complexes, the effects on activity were unidirectional, and in one complex (FbaB⋅PPase), the effects were reciprocal. The effects of potential physiological significance include inhibition of PPase by FbaB and GadA and activation of FbaB and cPGI by PPase. Together, they provide a mechanism for feedback regulation of FbaB and GadA biosynthesis. GENERAL SIGNIFICANCE: These findings indicate the complexity of functionally significant interactions between cellular enzymes, which classical enzymology treats as individual entities, and demonstrate their moonlighting activities as regulators.

Efficient production of γ-aminobutyric acid by glutamate decarboxylase immobilized on an amphiphilic organic-inorganic hybrid porous material.

A novel organic-inorganic hybrid porous material (KCS-2), containing both lipophilic and hydrophilic nanospaces to mimic a lipid bilayer, was utilized as an immobilization support and reaction accelerator for glutamate decarboxylase (GADß). Upon evaluation of the adsorption of GADß on KCS-2, the amount of immobilization was found to be approximately four times higher than that on non-porous silica, and a comparable adsorbability to mesoporous silica was observed. Following γ-aminobutyric acid (GABA) production by the decarboxylation of l-glutamic acid using these immobilized enzymes, the enzymatic activity of the GADß-KCS-2 composite was found to be significantly higher than that of the free enzyme. In contrast, the activity of the more common GADß-mesoporous silica composite decreased. Furthermore, the enzymatic activity of the GADß-KCS-2 composite was superior to those of the un-immobilized free enzyme and the amorphous material itself over a wide temperature range. Thereby, these findings suggest that the amphiphilic nanospace of KCS-2 is suitable as a stable enzyme immobilization field and reaction acceleration field under such conditions. In addition, the durability of the immobilized enzyme was examined in terms of GABA production, with approximately 20% activity retention being observed after 10 cycles using KCS-2. Such durability was not observed for the non-porous silica material due to enzyme desorption.

Development of a dual-fluorescence reporter system for high-throughput screening of L-aspartate-α-decarboxylase.

ß-Alanine (3-aminopropionic acid) holds great potential in industrial application. It can be obtained through a chemical synthesis route, which is hazardous to the environment. It is well known that l-aspartate-α-decarboxylase (ADC) can convert l-aspartate to ß-alanine in bacteria. However, due to the low activity of ADC, industrial production of ß-alanine through the green biological route remains unclear. Thus, improving the activity of ADC is critical to reduce the cost of ß-alanine production. In this study, we established a dual-fluorescence high-throughput system for efficient ADC screening. By measuring the amount of ß-alanine and the expression level of ADC using two different fluorescence markers, we can rapidly quantify the relative activity of ADC variants. From a mutagenesis library containing 2000 ADC variants, we obtained a mutant with 33% increased activity. Further analysis revealed that mutations of K43R and P103Q in ADC significantly improved the yield of ß-alanine produced by the whole-cell biocatalysis. Compared with the previous single-fluorescence method, our system can not only quantify the amount of ß-alanine but also measure the expression level of ADC with different fluorescence, making it able to effectively screen out ADC variants with improved relative activity. The dual-fluorescence high-throughput system for rapid screening of ADC provides a good strategy for industrial production of ß-alanine via the biological conversion route in the future.

Immobilization and enzymatic properties of glutamate decarboxylase from Enterococcus faecium by affinity adsorption on regenerated chitin.

Glutamate decarboxylase (GAD, EC 4.1.1.15) is an important enzyme in gamma-aminobutyric acid biosynthesis and DL-glutamic acid resolution. In this study, the Enterococcus faecium-derived GAD was successfully immobilized by regenerated chitin (RC) via specific adsorption of cellulose-binding domain (CBD). The optimal binding buffer was 20 mmol/L phosphate buffer saline (pH 8.0), and the RC binding capacity was 1.77 ± 0.11 mgcbd-gad/grc under this condition. The ratio of wet RC and crude enzyme solution used for immobilization was recommended to 3:50 (g/mL). To evaluate the effect of RC immobilization on GAD, properties of the immobilize GAD (RC-CBD-GAD) were investigated. Results indicated RC-CBD-GAD was relatively stable at pH 4.4-5.6 and temperature - 20-40 °C, and the optimal reaction pH value and temperature were pH 4.8 and 50 °C, respectively. When it was reacted with 5 mmol/L of follow chemical reagents respectively, the activity of RC-CBD-GAD was hardly affected by EDTA, KCl, and NaCl, and significantly inactivated by AgNO3, MnSO4, MgSO4, CuSO4, ZnSO4, FeCl2, FeCl3, AlCl3, CaCl2, and Pb(CH3COO)2. The apparent Km and Vmax were 28.35 mmol/L and 147.06 µmol/(gRC-CBD-GAD·min), respectively. The optimum time for a batch of catalytic reaction without exogenous pH control was 2 h. Under this reaction time, RC-CBD-GAD had a good reusability with a half-life of 23 cycles, indicating that it was very attractive for GABA industry. As a novel, efficient, and green CBD binding carrier, RC provides an alternative way to protein immobilization.

Protein Engineering of a Pyridoxal-5'-Phosphate-Dependent l-Aspartate-α-Decarboxylase from Tribolium castaneum for ß-Alanine Production.

In the present study, a pyridoxal-5'-phosphate (PLP)-dependent L-aspartate-α-decarboxylase from Tribolium castaneum (TcPanD) was selected for protein engineering to efficiently produce ß-alanine. A mutant TcPanD-R98H/K305S with a 2.45-fold higher activity than the wide type was selected through error-prone PCR, site-saturation mutagenesis, and 96-well plate screening technologies. The characterization of purified enzyme TcPanD-R98H/K305S showed that the optimal cofactor PLP concentration, temperature, and pH were 0.04% (m/v), 50 °C, and 7.0, respectively. The 1mM of Na+, Ni2+, Co2+, K+, and Ca2+ stimulated the activity of TcPanD-R98H/K305S, while only 5 mM of Ni2+ and Na+ could increase its activity. The kinetic analysis indicated that TcPanD-R98H/K305S had a higher substrate affinity and enzymatic reaction rate than the wild enzyme. A total of 267 g/L substrate l-aspartic acid was consumed and 170.5 g/L of ß-alanine with a molar conversion of 95.5% was obtained under the optimal condition and 5-L reactor fermentation.

Placental tissue of greenhouse muskmelon (Cucumis melo L.) contains more gamma-aminobutyric acid with antioxidant capacity than the fleshed pulp.

Our previous study revealed that gamma-aminobutyric acid (GABA) in Earl's muskmelon is more concentrated in the inner than the outer parts of the fruit. Here, the GABA and antioxidant capacity of the placental tissue of muskmelon, which is considered waste, were evaluated for possible use as a source of bioactive compounds. The concentrations of GABA and related substances in the placental tissue were significantly higher than in the fleshed pulp, whereas glutamic acid and sugar levels were significantly lower. The two sites showed no difference in GAD activity. Furthermore, the placental site showed high antioxidant capacities based on 2,2-diphenyl-1-picrylhydrazyl and oxygen radical absorbance capacity for hydrophilic compounds assays compared with the fleshed pulp, because of the higher levels of total phenolic and L-ascorbic acids. Therefore, the placental tissue of muskmelons may be useful for developing functional foods, which would also reduce the amount of residues during muskmelon processing.

Immune-mediated epilepsy with GAD65 antibodies.

Anti-GAD65 antibodies have been identified in both acute/subacute seizures (limbic encephalitis and extralimbic encephalitis) and chronic isolated epilepsy. The evidence of high serum titers and intrathecal synthesis play a fundamental role in diagnosis but poorly correlate with disease severity or response to therapies. It remains controversial whether anti-GAD65 Abs are the pathogenic entity or only serve as a surrogate marker for autoimmune disorders mediated by cytotoxic T cells. Unlike other immune-mediated epilepsy, although multiple combinations of therapeutics are used, the efficacy and prognosis of patients with GAD65-epilepsy patients are poor. Besides, GAD65-epilepsy is more prone to relapse and potentially evolve into a more widespread CNS inflammatory disorder. This article reviews the recent advances of GAD65-epilepsy, focusing on the diagnosis, epidemiology, pathophysiology, clinical features, and treatment, to better promote the recognition and provide proper therapy for this condition.

Engineering protonation conformation of l-aspartate-α-decarboxylase to relieve mechanism-based inactivation.

Mechanism-based inactivation of l-aspartate-α-decarboxylase (PanD), which leads to irreversible modification of active site, is a major challenge in the efficient production of ß-alanine from L-aspartic acid. In this study, a semi-rational strategy that combined conformational dynamics and structural alignment was applied to increase the catalytic stability of Bacillus subtilis PanD (BsPanD). Using site-saturation and C-terminal deletion, the variant Q5 (BsPanDI46V/I88M/K104S/I126* ) was generated. The catalytic half-life and the total turnover number (TTN) of Q5 were 3.48-fold and 2.52-fold higher, respectively, compared with that of the parent Q0. The reasons for the differences were the prolonged distance d1 between the phenolic group of Tyr58 and pyruvoyl group of Ser25 (4.9 Å in Q0 vs. 5.5 Å in Q5), an increased difficulty for incorrect protonation to occur, and the decreased flexibility of residues in regions A, B, and C, thereby enhancing the probability of correct protonation. Variant Q5, coupled with l-aspartase (AspA) in a 15-L bioreactor, generated a linear cascade system using fumaric acid as a substrate, yielding 118.6 g/L ß-alanine with a product/catalyst (P/C) ratio of 5.9 g/g and a conversion > 99%. These results showed that reshaping the protonation conformation of PanD can efficiently relieve mechanism-based inactivation and boost catalytic stability.

Fine Mapping of Glutamate Decarboxylase 65 Epitopes Reveals Dependency on Hydrophobic Amino Acids for Specific Interactions.

Characterization of multiple antibody epitopes has revealed the necessity of specific groups of amino acid residues for reactivity. This applies to the majority of antibody-antigen interactions, where especially charged and hydrophilic amino acids have been reported to be essential for antibody reactivity. This study describes thorough characterization of glutamic acid decarboxylase (GAD) 65 antigenic epitopes, an immunodominant autoantigen in type 1 diabetes (T1D). As linear epitopes are sparsely described for GAD65 in T1D, we aimed to identify and thoroughly characterize two GAD65 antibodies using immunoassays. A monoclonal antibody recognized an epitope in the N-terminal domain of GAD65, 8FWSFGSE14, whereas a polyclonal antibody recognized two continuous epitopes in the C-terminal domain, corresponding to amino acids 514RTLED518 and 549PLGDKVNF556. Hydrophobic amino acids were essential for antibody reactivity, which was verified by competitive inhibition assays. Moreover, the epitopes were located in flexible linker regions and turn structures. These findings confirm the versatile nature of antibody-antigen interactions and describe potential continuous epitopes related to T1D, which predominantly have been proposed to be of discontinuous nature.

Structural change in GadD2 of Listeria monocytogenes field isolates supports nisin resistance.

The lantibiotic nisin is used as a food additive to effectively inactivate a broad spectrum of Gram-positive bacteria such as Listeria monocytogenes. In total, 282 L. monocytogenes field isolates from German ready-to-eat food products, food-processing environments and patient samples and 39 Listeria reference strains were evaluated for their susceptibility to nisin. The MIC90 value was <1500 IU ml-1. Whole genome sequences (WGS) of four nisin susceptible (NS; growth <200 IU ml-1) and two nisin resistant L. monocytogenes field isolates (NR; growth >1500 IU ml-1) of serotype IIa were analyzed for DNA sequence variants (DSVs) in genes putatively associated with NR and its regulation. WGS of NR differed from NS in the gadD2 gene encoding for the glutamate decarboxylase system (GAD). Moreover, homology modeling predicted a protein structure of GadD2 in NR that promoted a less pH dependent GAD activity and may therefore be beneficial for nisin resistance. Likewise NR had a significant faster growth rate compared to NS in presence of nisin at pH 7. In conclusion, results contributed to ongoing debate that a genetic shift in GAD supports NR state.

Protein-engineered molecules carrying GAD65 epitopes and targeting CD35 selectively down-modulate disease-associated human B lymphocytes.

Type 1 diabetes mellitus is an autoimmune metabolic disorder characterized by chronic hyperglycemia, the presence of autoreactive T and B cells and autoantibodies against self-antigens. A membrane-bound enzyme on the pancreatic beta-cells, glutamic acid decarboxylase 65 (GAD65), is one of the main autoantigens in type 1 diabetes. Autoantibodies against GAD65 are potentially involved in beta-cell destruction and decline of pancreatic functions. The human complement receptor type 1 (CD35) on B and T lymphocytes has a suppressive activity on these cells. We hypothesized that it may be possible to eliminate GAD65-specific B cells from type 1 diabetes patients by using chimeric molecules, containing an anti-CD35 antibody, coupled to peptides resembling GAD65 B/T epitopes. These molecules are expected to selectively bind the anti-GAD65 specific B cells by the co-cross-linking of the immunoglobulin receptor and CD35 and to deliver a suppressive signal. Two synthetic peptides derived from GAD65 protein (GAD65 epitopes) and anti-CD35 monoclonal antibody were used for the construction of two chimeras. The immunomodulatory activity of the engineered antibodies was tested in vitro using peripheral blood mononuclear cells (PBMCs) from type 1 diabetes patients. A reduction in the number of anti-GAD65 IgG antibody-secreting plasma cells and increased percentage of apoptotic B lymphocytes was observed after treatment of these PBMCs with the engineered antibodies. The constructed chimeric molecules are able to selectively modulate the activity of GAD65-specific B lymphocytes and the production of anti-GAD65 IgG autoantibodies by co-cross-linking of the inhibitory CD35 and the B cell antigen receptor (BCR). This treatment presents a possible way to alter the autoimmune nature of these cells.

The intervention effect of licorice in d-galactose induced aging rats by regulating the taurine metabolic pathway.

Licorice, an edible and officinal plant material, has attracted considerable attention for its wide range of pharmacological activities. Our previous study showed that licorice can ameliorate cognitive damage and improve oxidative stress and apoptosis in aging rats induced by d-galactose (d-gal). In this study, in order to further explore the changes of the metabolic profile during the aging process and the antiaging mechanism of licorice, the 1H NMR-based metabolomics approach was used to analyze serum and urine samples and identify a potential biomarker in d-gal induced aging rats. The results revealed that the taurine metabolic pathway was significantly correlated with the ageing process in d-gal induced rats. Furthermore, the taurine contents were significantly decreased in both the serum and urine samples of aging rats compared with the controls. At the same time, the levels of cysteine dioxygenase type I (CDO1), cysteine sulfinic acid decarboxylase (CSAD) and glutamate decarboxylase type I (GAD1), which are the key enzymes affecting the synthesis reactions, were decreased in aging rats compared with the controls. After licorice administration, the levels of taurine, CDO1 and CSAD were all significantly increased. These findings firstly demonstrated that the regulation of the taurine metabolic pathway is involved in the anti-aging effect of licorice in d-gal induced aging rats.

Identification of mutations restricting autocatalytic activation of bacterial L-aspartate α-decarboxylase.

Bacterial L-aspartate α-decarboxylase (PanD) specifically catalyzes the decarboxylation of L-aspartic acid to ß-alanine. It is translated as an inactive pro-protein, then processed by self-cleavage to form two small subunits with catalytic activity. There is a significant difference in the efficiency of this process among the reported PanDs, while the structural basis remains unclear. More PanDs with known sequences and characterized properties are needed to shed light on the molecular basis of the self-cleavage process. In this study, PanD genes from 33 selected origins were synthesized and expressed; using purified recombinant enzymes, their self-processing properties were characterized and classified. Three classes of PanDs were acquired based on their self-cleavage efficiency. Combined with the phylogenetic analysis and structure comparison, sited-directed mutagenesis was performed to investigate the effects of four mutants on self-processing. In comparison with the wild-type (96.4%), the self-cleavage efficiencies of mutants V23E, I26C, T27A, and E56S were decreased to 90.5, 83.6, 74.4 and 81.2%, respectively. The results indicated that residues of V23, I26, T27 and E56 were critical to the self-cleavage processing of PanDs. This work provided further understanding to the self-cleavage processing of PanDs, which may contribute to protein engineering of the enzyme.

Lactobacillus brevis CGMCC 1306 glutamate decarboxylase: Crystal structure and functional analysis.

Glutamate decarboxylase (GAD), which is a unique pyridoxal 5-phosphate (PLP)-dependent enzyme, can catalyze α-decarboxylation of l-glutamate (L-Glu) to γ-aminobutyrate (GABA). The crystal structure of GAD in complex with PLP from Lactobacillus brevis CGMCC 1306 was successfully solved by molecular-replacement, and refined at 2.2 Šresolution to an Rwork factor of 18.76% (Rfree = 23.08%). The coenzyme pyridoxal 5-phosphate (PLP) forms a Schiff base with the active-site residue Lys279 by continuous electron density map, which is critical for catalysis by PLP-dependent decarboxylase. Gel filtration showed that the active (pH 4.8) and inactive (pH 7.0) forms of GAD are all dimer. The residues (Ser126, Ser127, Cys168, Ile211, Ser276, His278 and Ser321) play important roles in anchoring PLP cofactor inside the active site and supporting its catalytic reactivity. The mutant T215A around the putative substrate pocket displayed an 1.6-fold improvement in catalytic efficiency (kcat/Km) compared to the wild-type enzyme (1.227 mM-1 S-1 versus 0.777 mM-1 S-1), which was the highest activity among all variants tested. The flexible loop (Tyr308-Glu312), which is positioned near the substrate-binding site, is involved in the catalytic reaction, and the conserved residue Tyr308 plays a vital role in decarboxylation of L-Glu.

Increasing thermal stability of glutamate decarboxylase from Escherichia. coli by site-directed saturation mutagenesis and its application in GABA production.

Gamma-amino butyric acid (GABA) is an important bio-product used in pharmaceuticals, functional foods, and a precursor of the biodegradable plastic polyamide 4 (Nylon 4). Glutamate decarboxylase B (GadB) from Escherichia. coli is a highly active biocatalyst that can convert l-glutamate to GABA. However, its practical application is limited by the poor thermostability and only active under acidic conditions of GadB. In this study, we performed site-directed saturation mutagenesis of the N-terminal residues of GadB from Escherichia coli to improve its thermostability. A triple mutant (M6, Gln5Ile/Val6Asp/Thr7Gln) showed higher thermostability, with a 5.6 times (560%) increase in half-life value at 45 °C, 8.7 °C rise in melting temperature (Tm) and a 14.3 °C rise in the temperature at which 50% of the initial activity remained after 15 min incubation (T1550), compared to wild-type enzyme. Protein 3D structure analysis showed that the induced new hydrogen bonds in the same polypeptide chain or between polypeptide chains in E. coli GadB homo-hexamer may be responsible for the improved thermostability. Increased thermostability contributed to increased GABA conversion ability. After 12 h conversion of 3 mol/L l-glutamate, GABA produced and mole conversion rate catalyzed by M6 whole cells was 297 g/L and 95%, respectively, while those by wild-type GAD was 273.5 g/L and 86.2%, respectively.

Elements of molecular machinery of GABAergic signaling in the vertebrate cholinergic neuromuscular junction.

It is generally accepted that gamma-aminobutyric acid (GABA) is a signaling molecule abundant in central synapses. In a number of studies though, it has been shown that GABA signaling functions in the peripheral nervous system as well, in particular, in the synapses of sympathetic ganglia. However, there exists no firm evidence on the presence of GABAergic signaling cascade in the intercellular junctions of the somatic nerve system. By the use of immunohistochemistry methods, in the synaptic area of cholinergic neuromuscular contact in rat diaphragm, we have detected glutamate decarboxylase, the enzyme involved in synthesis of GABA, molecules of GABA, and also GAT-2, a protein responsible for transmembrane transport of GABA. Earlier we have also shown that metabotropic GABAB receptors have overlapping localization in the same compartment. Moreover, activation of GABAB receptors affects the intensity of acetylcholine release. These data taken together, allows us to suggest that in the mammalian cholinergic neuromuscular junction, GABA is synthesized and performs certain synaptic signaling function.