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66-423: In mammals, GAD exists in two isoforms with molecular weights of 67 and 65 kDa (GAD 67 and GAD 65 ), which are encoded by two different genes on different chromosomes ( GAD1 and GAD2 genes, chromosomes 2 and 10 in humans, respectively). GAD 67 and GAD 65 are expressed in the brain where GABA is used as a neurotransmitter , and they are also expressed in the insulin -producing β-cells of

132-416: A coenzyme in all transamination reactions, and in certain decarboxylation , deamination , and racemization reactions of amino acids . The aldehyde group of PLP forms a Schiff-base linkage (internal aldimine ) with the ε-amino group of a specific lysine group of the aminotransferase enzyme. The α-amino group of the amino acid substrate displaces the ε-amino group of the active-site lysine residue in

198-963: A comparison of autistic versus control brains, GAD65 and GAD67 experienced a downregulation average of 50% in parietal and cerebellar cortices of autistic brains. Cerebellar Purkinje cells also reported a 40% downregulation, suggesting that affected cerebellar nuclei may disrupt output to higher order motor and cognitive areas of the brain. Both GAD 67 and GAD 65 are targets of autoantibodies in people who later develop type 1 diabetes mellitus or latent autoimmune diabetes . Injections with GAD 65 in ways that induce immune tolerance have been shown to prevent type 1 diabetes in rodent models. In clinical trials, injections with GAD 65 have been shown to preserve some insulin production for 30 months in humans with type 1 diabetes. A Cochrane systematic review also examined 1 study showing improvement of C-peptide levels in cases of Latent Autoimmune Diabetes in adults, 5 years following treatment with GAD 65 .Still, it

264-434: A gene that serves as an initial binding site—resulting in slightly modified transcripts and protein isoforms. Generally, one protein isoform is labeled as the canonical sequence based on criteria such as its prevalence and similarity to orthologous —or functionally analogous—sequences in other species. Isoforms are assumed to have similar functional properties, as most have similar sequences, and share some to most exons with

330-479: A major role in conferring acid resistance and allows bacteria to temporarily survive in highly acidic environments (pH < 2.5) like the stomach. This is done by GAD decarboxylating glutamate to GABA, which requires H+ to be uptaken as a reactant and raises the pH inside the bacteria. GABA can then be exported out of E. coli cells and contribute to increasing the pH of the nearby extracellular environments. Isoform A protein isoform , or " protein variant ",

396-497: A phosphoric acid. Several inhibitors of PLP enzymes are known. One type of inhibitor forms an electrophile with PLP, causing it to irreversibly react with the active site lysine. Acetylenic compounds (e.g. propargylglycine) and vinylic compounds (e.g. vinylglycine) are such inhibitors. A different type of inhibitor inactivates PLP, and such are α-methyl and amino-oxy substrate analogs (e.g. α-methylglutamate). Still other inhibitors have good leaving groups that nucleophilically attack

462-414: A process known as transaldimination. The resulting external aldimine can lose a proton, carbon dioxide, or an amino acid sidechain to become a quinonoid intermediate, which in turn can act as a nucleophile in several reaction pathways. In transamination, after deprotonation the quinonoid intermediate accepts a proton at a different position to become a ketimine . The resulting ketimine is hydrolysed so that

528-467: A step catalyzed by PLP-synthase, an enzyme composed of two subunits. PdxS catalyzes the condensation of ribulose 5-phosphate, glyceraldehyde-3-phosphate, and ammonia , this latter molecules is produced by PdxT which catalyzes the production of ammonia from glutamine . PdxS is a (β/α)8 barrel (also known as a TIM-barrel) that forms a dodecamer. The widespread utilization of PLP in central metabolism, especially in amino acid biosynthesis, and its activity in

594-461: Is pyridoxine 5'-phosphate . The condensation is catalyzed by PNP synthase , encoded by pdxJ , which creates PNP (pyridoxine 5' phosphate). The final enzyme is PNP oxidase ( pdxH ), which catalyzes the oxidation of the 4' hydroxyl group to an aldehyde using dioxigen, resulting in hydrogen peroxide. The first branch is catalyzed in E. coli by enzymes encoded by epd , pdxB , serC and pdxA . These share mechanistical similarities and homology with

660-421: Is a key enzyme involved in the synthesis of inhibitory neurotransmitter GABA and people with schizophrenia have been shown to express lower amounts of GAD 67 in the dorsolateral prefrontal cortex compared to healthy controls. The mechanism underlying the decreased levels of GAD 67 in people with schizophrenia remains unclear. Some have proposed that an immediate early gene, Zif268, which normally binds to

726-526: Is a member of a set of highly similar proteins that originate from a single gene and are the result of genetic differences. While many perform the same or similar biological roles, some isoforms have unique functions. A set of protein isoforms may be formed from alternative splicings , variable promoter usage, or other post-transcriptional modifications of a single gene; post-translational modifications are generally not considered. (For that, see Proteoforms .) Through RNA splicing mechanisms, mRNA has

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792-436: Is a precursor to polyamines. Pyridoxal phosphate has numerous roles in human body. A few examples below: PLP is also found on glycogen phosphorylase in the liver, where it is used to break down glycogen in glycogenolysis when glucagon or epinephrine signals it to do so. However, this enzyme does not exploit the reactive aldehyde group, but instead utilizes the phosphate group on PLP to perform its reaction. Although

858-451: Is activated when bound to PLP and inactive when not bound to PLP. Majority of GAD 67 is bound to PLP at any given time, whereas GAD 65 binds PLP when GABA is needed for neurotransmission. This reflects the functional properties of the two isoforms; GAD 67 must be active at all times for normal cellular functioning, and is therefore constantly activated by PLP, while GAD 65 must only be activated when GABA neurotransmission occurs, and

924-492: Is an example of such an enzyme. Human Serine hydroxymethyltransferase 2 regulates one-carbon transfer reactions required for amino acid and nucleotide metabolism, and exists in dimeric and tetrameric forms. The dimeric SHMT2 variant is a potent inhibitor of the BRISC deubiquitylase enzyme complex, which regulates immune-based cell signaling. Recent studies show that SJMT2 tetramerization is induced by PLP. This prevents interaction with

990-466: Is generated, commonly referred to as the external aldimine. After this step, the pathway for each PLP-catalyzed reactions diverge. Specificity is conferred by the fact that, of the four bonds of the alpha-carbon of the amino acid aldimine state, the bond perpendicular to the pyridine ring will be broken ( Dunathan Stereoelectronic Hypothesis ). Consequently, specificity is dictated by how the enzymes bind their substrates. An additional role in specificity

1056-446: Is important to highlight that the studies available to be included in this review presented considerable flaws in quality and design. High titers of autoantibodies to glutamic acid decarboxylase (GAD) are well documented in association with stiff person syndrome (SPS). Glutamic acid decarboxylase is the rate-limiting enzyme in the synthesis of γ-aminobutyric acid (GABA), and impaired function of GABAergic neurons has been implicated in

1122-591: Is mediated by a short flexible loop at the dimer interface (residues 432–442 in GAD67, and 423–433 in GAD65). In GAD67 this loop remains tethered, covering the active site and providing a catalytic environment to sustain GABA production; its mobility in GAD65 promotes a side reaction that results in release of PLP, leading to autoinactivation. The conformation of this loop is intimately linked to

1188-532: Is no conclusive evidence that it acts primarily by producing novel protein isoforms. Alternative splicing generally describes a tightly regulated process in which alternative transcripts are intentionally generated by the splicing machinery. However, such transcripts are also produced by splicing errors in a process called "noisy splicing," and are also potentially translated into protein isoforms. Although ~95% of multi-exonic genes are thought to be alternatively spliced, one study on noisy splicing observed that most of

1254-543: Is often used as a proxy for the abundance of protein isoforms, though proteomics experiments using gel electrophoresis and mass spectrometry have demonstrated that the correlation between transcript and protein counts is often low, and that one protein isoform is usually dominant. One 2015 study states that the cause of this discrepancy likely occurs after translation, though the mechanism is essentially unknown. Consequently, although alternative splicing has been implicated as an important link between variation and disease, there

1320-532: Is only necessary at nerve terminals and synapses. In order to aid in neurotransmission, GAD 65 forms a complex with heat shock cognate 70 (HSC 70 ), cysteine string protein (CSP) and vesicular GABA transporter VGAT, which, as a complex, helps package GABA into vesicles for release during neurotransmission. GAD 67 is transcribed during early development, while GAD 65 is not transcribed until later in life. This developmental difference in GAD 67 and GAD 65 reflects

1386-413: Is played by the ease of protonation of the pyridine ring nitrogen. PLP is retained in the active site not only thanks to the lysine, but also thanks to the interaction of the phosphate group and a phosphate binding pocket and to a lesser extent thanks to base stacking of the pyridine ring with an overhanging aromatic residue, generally tyrosine (which may also partake in the acid–base catalysis). Despite

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1452-568: Is positioned such that its carboxyl group forms a salt bridge with arginine and a hydrogen bond with glutamine. Dimerization is essential to maintaining function as the active site is found at this interface, and mutations interfering with optimal association between the 2 chains has been linked to pathology, such as schizophrenia. Interference of dimerization by GAD inhibitors such as 2-keto-4-pentenoic acid (KPA) and ethyl ketopentenoate (EKP) were also shown to lead to dramatic reductions in GABA production and incidence of seizures. Catalytic activity

1518-474: Is synthesized from pyridoxal by the enzyme pyridoxal kinase , requiring one ATP molecule. PLP is metabolized in the liver. Two natural pathways for PLP are currently known: one requires deoxyxylulose 5-phosphate (DXP), while the other does not, hence they are known as DXP-dependent and DXP-independent. These pathways have been studied extensively in Escherichia coli and Bacillus subtilis , respectively. Despite

1584-692: Is the main post-transcriptional modification process that produces mRNA transcript isoforms, and is a major molecular mechanism that may contribute to protein diversity. The spliceosome , a large ribonucleoprotein , is the molecular machine inside the nucleus responsible for RNA cleavage and ligation , removing non-protein coding segments ( introns ). Because splicing is a process that occurs between transcription and translation , its primary effects have mainly been studied through genomics techniques—for example, microarray analyses and RNA sequencing have been used to identify alternatively spliced transcripts and measure their abundances. Transcript abundance

1650-475: Is therefore regulated according to the synaptic environment. Studies with mice also show functional differences between Gad67 and Gad65. GAD67−/− mice are born with cleft palate and die within a day after birth while GAD65−/− mice survive with a slightly increased tendency in seizures. Additionally, GAD65+/- have symptoms defined similarly to attention deficit hyperactivity disorder (ADHD) in humans. Both GAD67 and GAD65 are present in all types of synapses within

1716-507: The 5' AMP-activated protein kinase (AMPK), an enzyme, which performs different roles in human cells, has 3 subunits: In human skeletal muscle, the preferred form is α2β2γ1. But in the human liver, the most abundant form is α1β2γ1. The primary mechanisms that produce protein isoforms are alternative splicing and variable promoter usage, though modifications due to genetic changes, such as mutations and polymorphisms are sometimes also considered distinct isoforms. Alternative splicing

1782-416: The blood proteins as orosomucoid , antitrypsin , and haptoglobin . An unusual glycoform variation is seen in neuronal cell adhesion molecule, NCAM involving polysialic acids, PSA . Monoamine oxidase , a family of enzymes that catalyze the oxidation of monoamines, exists in two isoforms, MAO-A and MAO-B. Pyridoxal phosphate Pyridoxal phosphate ( PLP , pyridoxal 5'- phosphate , P5P ),

1848-407: The pancreas , in varying ratios depending upon the species. Together, these two enzymes maintain the major physiological supply of GABA in mammals, though it may also be synthesized from putrescine in the enteric nervous system , brain, and elsewhere by the actions of diamine oxidase and aldehyde dehydrogenase 1a1 . Several truncated transcripts and polypeptides of GAD 67 are detectable in

1914-488: The promoter region of GAD 67 and increases transcription of GAD 67 , is lower in schizophrenic patients, thus contributing to decreased levels of GAD 67 . Since the dorsolateral prefrontal cortex (DLPFC) is involved in working memory, and GAD 67 and Zif268 mRNA levels are lower in the DLPFC of schizophrenic patients, this molecular alteration may account, at least in part, for the working memory impairments associated with

1980-460: The BRISC deubiqutylase complex, potentially linking vitamin B6 levels and metabolism to inflammation. The pyridoxal-5′-phosphate-dependent enzymes (PLP enzymes) catalyze myriad reactions. Although the scope of PLP-catalyzed reactions appears to be immense, the unifying principle is the formation of an internal lysine-derived aldimine. Once the amino substrate interacts with the active site, a new Schiff base

2046-504: The C-terminal domain, which also affects the rate of autoinactivation. Moreover, GABA-bound GAD65 is intrinsically more flexible and exists as an ensemble of states, thus providing more opportunities for autoantigenicity as seen in Type 1 diabetes. GAD derived from Escherichia coli shows additional structural intricacies, including a pH-dependent conformational change. This behavior is defined by

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2112-506: The CaMBD, preventing the protein from binding to GAD. Whereas CaMBD of the isoform in rice still functions as an autoinhibitory domain, the C-terminus in the isoform in apples does not. Finally, the structure of plant GAD is a hexamer and has pH-dependent activity, with the optimal pH of 5.8 in multiple species. but also significant activity at pH 7.3 in the presence of CaM It is also believed that

2178-672: The GABA produced by GAD is used in plants to signal abiotic stress by controlling levels of intracellular Ca via CaM. Binding to CaM opens Ca channels and leads to an increase in Ca concentrations in the cytosol, allowing Ca to act as a secondary messenger and activate downstream pathways. When GAD is not bound to CaM, the CaMBD acts as an autoinhibitory domain, thus deactivating GAD in the absence of stress. Interesting, in two plant species, rice and apples, Ca2+ /CAM-independent GAD isoforms have been discovered. The C-terminus of these isoforms contain substitutions at key residues necessary to interact with CaM in

2244-525: The PLP. Such is chloroalanine , which inhibits a large number of enzymes. Examples of inhibitors: Pyridoxal-5-phosphate (vitamin B6) -dependent enzymes have multiple evolutionary origins. The overall B6 enzymes diverged into four independent evolutionary lines: α family (i.e. aspartate aminotransferase ), β family ( serine dehydratase ),D- alanine aminotransferase family and the alanine racemase family. An example of

2310-456: The ability to select different protein-coding segments ( exons ) of a gene, or even different parts of exons from RNA to form different mRNA sequences. Each unique sequence produces a specific form of a protein. The discovery of isoforms could explain the discrepancy between the small number of protein coding regions of genes revealed by the human genome project and the large diversity of proteins seen in an organism: different proteins encoded by

2376-457: The absence of enzymes, suggests PLP may be a "prebiotic" compound—that is, one that predates the origin of organic life (not to be confused with prebiotic compounds , substances which serve as a food source for beneficial bacteria). In fact, heating NH3 and Glycolaldehyde spontaneously forms a variety of pyridines, including pyridoxal. Under certain conditions, PLP is formed from cyanoacetylene, diacetylene, carbon monoxide, hydrogen, water, and

2442-478: The active form of vitamin B 6 , is a coenzyme in a variety of enzymatic reactions. The International Union of Biochemistry and Molecular Biology has catalogued more than 140 PLP-dependent activities, corresponding to ~4% of all classified activities. The versatility of PLP arises from its ability to covalently bind the substrate, and then to act as an electrophilic catalyst, thereby stabilizing different types of carbanionic reaction intermediates. PLP acts as

2508-451: The amino group remains on the complex. In addition, PLP is used by aminotransferases (or transaminases) that act upon unusual sugars such as perosamine and desosamine . In these reactions, the PLP reacts with glutamate , which transfers its alpha-amino group to PLP to make pyridoxamine phosphate (PMP). PMP then transfers its nitrogen to the sugar, making an amino sugar . PLP is also involved in various beta-elimination reactions such as

2574-491: The canonical sequence. However, some isoforms show much greater divergence (for example, through trans-splicing ), and can share few to no exons with the canonical sequence. In addition, they can have different biological effects—for example, in an extreme case, the function of one isoform can promote cell survival, while another promotes cell death—or can have similar basic functions but differ in their sub-cellular localization. A 2016 study, however, functionally characterized all

2640-456: The cell, at different developmental times, and for functionally different purposes. GAD 67 is spread evenly throughout the cell while GAD 65 is localized to nerve terminals. GAD 67 synthesizes GABA for neuron activity unrelated to neurotransmission, such as synaptogenesis and protection from neural injury. This function requires widespread, ubiquitous presence of GABA. GAD 65 , however, synthesizes GABA for neurotransmission, and therefore

2706-400: The control of glutamate decarboxylase has the prospect of improving citrus produce quality post-harvest. In Citrus plants, research has shown that glutamate decarboxylase plays a key role in citrate metabolism. With the increase of glutamate decarboxylase via direct exposure, citrate levels have been seen to significantly increase within plants, and in conjunction post-harvest quality maintenance

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2772-481: The developing brain, however their function, if any, is unknown. Both isoforms of GAD are homodimeric structures, consisting of three primary domains: the PLP, C-terminal and N-terminal domains. The PLP-binding domain of this enzyme adopts a type I PLP-dependent transferase-like fold. The reaction proceeds via the canonical mechanism, involving Schiff base linkage between PLP and Lys405. PLP is held in place through base-stacking with an adjacent histidine residue, and GABA

2838-486: The different low-abundance transcripts are noise, and predicts that most alternative transcript and protein isoforms present in a cell are not functionally relevant. Other transcriptional and post-transcriptional regulatory steps can also produce different protein isoforms. Variable promoter usage occurs when the transcriptional machinery of a cell ( RNA polymerase , transcription factors , and other enzymes ) begin transcription at different promoters—the region of DNA near

2904-420: The disease. The bilateral delivery of glutamic acid decarboxylase (GAD) by an adeno-associated viral vector into the subthalamic nucleus of patients between 30 and 75 years of age with advanced, progressive, levodopa-responsive Parkinson disease resulted in significant improvement over baseline during the course of a six-month study. Intracerebellar administration of GAD autoantibodies to animals increases

2970-535: The disparity in the starting compounds and the different number of steps required, the two pathways possess many commonalities. The DXP-dependent biosynthetic route requires several steps and a convergence of two branches, one producing 3-hydroxy-1-aminoacetone phosphate from erythrose 4-phosphate , while the other (single enzyme) producing deoxyxylulose 5-phosphate (DXP) from glyceraldehyde 3-phosphate (GAP) and pyruvate . The condensation product of 3-hydroxy-1-aminoacetone phosphate and deoxyxylulose 5-phosphate

3036-434: The excitability of motoneurons and impairs the production of nitric oxide (NO), a molecule involved in learning. Epitope recognition contributes to cerebellar involvement. Reduced GABA levels increase glutamate levels as a consequence of lower inhibition of subtypes of GABA receptors. Higher glutamate levels activate microglia and activation of xc(−) increases the extracellular glutamate release. Peripheral nerve injury of

3102-464: The function of each isoform must generally be determined separately, most identified and predicted isoforms still have unknown functions. A glycoform is an isoform of a protein that differs only with respect to the number or type of attached glycan . Glycoproteins often consist of a number of different glycoforms, with alterations in the attached saccharide or oligosaccharide . These modifications may result from differences in biosynthesis during

3168-403: The functional properties of each isoform; GAD 67 is needed throughout development for normal cellular functioning, while GAD 65 is not needed until slightly later in development when synaptic inhibition is more prevalent. GAD 67 and GAD 65 are also regulated differently post-translationally. Both GAD 65 and GAD 67 are regulated via phosphorylation of a dynamic catalytic loop, but

3234-402: The human nervous system. This includes dendrodendritic , axosomatic, and axodendritic synapses. Preliminary evidence suggests that GAD65 is dominant in the visual and neuroendocrine systems, which undergo more phasic changes. It is also believed that GAD67 is present at higher amounts in tonically active neurons. Both GAD65 and GAD67 experience significant downregulation in cases of autism . In

3300-413: The isoforms of 1,492 genes and determined that most isoforms behave as "functional alloforms." The authors came to the conclusion that isoforms behave like distinct proteins after observing that the functional of most isoforms did not overlap. Because the study was conducted on cells in vitro , it is not known if the isoforms in the expressed human proteome share these characteristics. Additionally, because

3366-446: The limited requirements for a PLP binding pocket, PLP enzymes belong to only five different families. These families do not correlate well with a particular type of reaction. The five families are classified as fold types followed by a Roman numeral. Animals are auxotroph for this enzyme co-factor and require it or an intermediate to be supplemented, hence its classification as a vitamin B 6 , unlike MoCo or CoQ10 for example. PLP

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3432-443: The pathogenesis of SPS. Autoantibodies to GAD might be the causative agent or a disease marker. Substantial dysregulation of GAD mRNA expression, coupled with downregulation of reelin , is observed in schizophrenia and bipolar disorder . The most pronounced downregulation of GAD 67 was found in hippocampal stratum oriens layer in both disorders and in other layers and structures of hippocampus with varying degrees. GAD 67

3498-478: The presence of a triple helical bundle formed by the N-termini of the hexameric protein in acidic environments. Despite an extensive sequence similarity between the two genes, GAD65 and GAD67 fulfill very different roles within the human body. Additionally, research suggests that GAD65 and GAD67 are regulated by distinctly different cellular mechanisms. GAD 65 and GAD 67 synthesize GABA at different locations in

3564-404: The process of glycosylation , or due to the action of glycosidases or glycosyltransferases . Glycoforms may be detected through detailed chemical analysis of separated glycoforms, but more conveniently detected through differential reaction with lectins , as in lectin affinity chromatography and lectin affinity electrophoresis . Typical examples of glycoproteins consisting of glycoforms are

3630-424: The reactions carried out by serine dehydratase and GDP-4-keto-6-deoxymannose-3-dehydratase (ColD) . It is also active in the condensation reaction in heme synthesis. PLP plays a role in the conversion of levodopa into dopamine , facilitates the conversion of the excitatory neurotransmitter glutamate to the inhibitory neurotransmitter GABA , and allows SAM to be decarboxylated to form propylamine , which

3696-502: The regulation of these isoforms differs; GAD 65 is activated by phosphorylation while GAD 67 is inhibited by phosphorylation. GAD67 is predominantly found activated (~92%), whereas GAD65 is predominantly found inactivated (~72%). GAD 67 is phosphorylated at threonine 91 by protein kinase A (PKA), while GAD 65 is phosphorylated, and therefore regulated by, protein kinase C (PKC). Both GAD 67 and GAD 65 are also regulated post-translationally by pyridoxal 5’-phosphate (PLP); GAD

3762-469: The same gene could increase the diversity of the proteome . Isoforms at the RNA level are readily characterized by cDNA transcript studies. Many human genes possess confirmed alternative splicing isoforms. It has been estimated that ~100,000 expressed sequence tags ( ESTs ) can be identified in humans. Isoforms at the protein level can manifest in the deletion of whole domains or shorter loops, usually located on

3828-671: The sciatic nerve (a neuropathic pain model) induces a transient loss of GAD 65 immunoreactive terminals in the spinal cord dorsal horn and suggests a potential involvement for these alterations in the development and amelioration of pain behaviour. Antibodies directed against glutamic acid decarboxylase (GAD) are increasingly found in patients with other symptoms indicative of central nervous system (CNS) dysfunction, such as ataxia , progressive encephalomyelitis with rigidity and myoclonus (PERM), limbic encephalitis , and epilepsy . The pattern of anti-GAD antibodies in epilepsy differs from type 1 diabetes and stiff-person syndrome. Besides

3894-438: The structure of most isoforms in the human proteome has been predicted by AlphaFold and publicly released at isoform.io . The specificity of translated isoforms is derived by the protein's structure/function, as well as the cell type and developmental stage during which they are produced. Determining specificity becomes more complicated when a protein has multiple subunits and each subunit has multiple isoforms. For example,

3960-565: The surface of the protein. One single gene has the ability to produce multiple proteins that differ both in structure and composition; this process is regulated by the alternative splicing of mRNA, though it is not clear to what extent such a process affects the diversity of the human proteome, as the abundance of mRNA transcript isoforms does not necessarily correlate with the abundance of protein isoforms. Three-dimensional protein structure comparisons can be used to help determine which, if any, isoforms represent functional protein products, and

4026-482: The synthesis of GABA, GAD has additional functions and structural variations that are organism-dependent. In Saccharomyces cerevisiae , GAD binds the Ca regulatory protein calmodulin (CaM) and is also involved in responding to oxidative stress. Similarly, GAD in plants binds calmodulin as well. This interaction occurs at the 30-50bp CAM-binding domain (CaMBD) in its C terminus and is necessary for proper regulation of GABA production. Unlike vertebrates and invertebrates,

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4092-409: The three enzymes in serine biosynthesis ( serA (homologue of pdxB ), serC , serB — however, epd is a homologue of gap ), which points towards a shared evolutionary origin of the two pathways. In several species there are two homologues of the E. coli serC gene, generally one in a ser operon ( serC ), and the other in a pdx operon, in which case it is called pdxF . A "serendipitous pathway"

4158-415: The vast majority of PLP-dependent enzymes form an internal aldimine with PLP via an active site lysine residue, some PLP-dependent enzymes do not have this lysine residue, but instead have a histidine in the active site. In such a case, the histidine cannot form the internal aldimine, and, therefore, the co-factor does not become covalently tethered to the enzyme. GDP-4-keto-6-deoxymannose-3-dehydratase (ColD)

4224-408: Was dephosphorylated, resulting in an unstable intermediate that decarboxylates spontaneously (hence the presence of the phosphate in the serine biosynthetic pathway) to glycaldehyde. Glycaldehyde was condensed with glycine and the phosphorylated product was 4-phosphohydroxythreonine (4PHT), the canonical substrate for 4-PHT dehydrogenase ( pdxA ). The DXP-independent PLP-biosynthetic route consists of

4290-443: Was found in an overexpression library that could suppress the auxotrophy caused by the deletion of pdxB (encoding erythronate 4 phosphate dehydrogenase) in E. coli . The serendipitous pathway was very inefficient, but was possible due to the promiscuous activity of various enzymes. It started with 3-phosphohydroxypyruvate (the product of the serA -encoded enzyme in serine biosynthesis) and did not require erythronate-4-phosphate. 3PHP

4356-468: Was significantly improved, and rot rates decreased. Just like GAD in plants, GAD in E. coli has a hexamer structure and is more active under acidic pH; the pH optimum for E. coli GAD is 3.8-4.6. However, unlike plants and yeast, GAD in E. coli does not require calmodulin binding to function. There are also two isoforms of GAD, namely GadA and GadB, encoded by separate genes in E. coli , although both isoforms are biochemically identical. The enzyme plays

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