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4 Aminobutyrate Aminotransferase

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Jorge E. Churchich – One of the best experts on this subject based on the ideXlab platform.

  • Gabaculine and m-carboxyphenyl-pyridoxamine 5-phosphate as probes of the catalytic binding sites of 4Aminobutyrate Aminotransferase.
    European journal of biochemistry, 2005
    Co-Authors: Doo Sik Kim, Udoudo Moses, Jorge E. Churchich

    Abstract:

    A homogeneous 4Aminobutyrate Aminotransferase isolated from pig brain exhibits a kcat of 9.6 s−1 and contains one mole of pyridoxal 5-phosphate/mole of dimer. The reaction of the enzyme with gabaculine (5-amino-1, 3-cyclohexadiene carboxylic acid) was studied by observing changes in the absorption spectrum of the bound cofactor and by monitoring loss of catalytic activity. The enzyme is completely inactivated by gabaculine, but the dialyzed inactive sample containing 0.5 mol of gabaculine/mol dimer is fully reconstituted by addition of pyridoxal 5-phosphate. Stopped-flow kinetic studies reveal that gabaculine reacts with the cofactor bound to the Aminotransferase with a second-order rate constant of 2.5 × 103 M−1s−1. Fluorometric titrations of the apoprotein with m-carboxyphenyl-pyridoxamine 5-phosphate show the binding of two moles of inhibitor/mole of enzyme. The binding process is reversible and the affinity of the apoprotein for the inhibitor is at least 10-fold higher than the affinity for the cofactor. It is postulated that the dimeric enzyme contains two potential active sites per dimer, but the binding site characterized by a weaker affinity constant for pyridoxal 5-phosphate becomes functional only after specific chemical modification of the molecule of cofactor tightly bound to the protein.

  • Recombinant brain 4Aminobutyrate Aminotransferases overexpression, purification, and identification of Lys-330 at the active site
    Biochimica et biophysica acta, 1997
    Co-Authors: Young Tae Kim, Young Hwan Song, Jorge E. Churchich

    Abstract:

    4Aminobutyrate Aminotransferase (4Aminobutyrate: 2-oxoglutarate Aminotransferase EC 2.6.1.19) is a key enzyme of the 4-aminobutyric acid shunt. It catalyzes the conversion of 4Aminobutyrate to succinic semialdehyde. In an effort to clarify the structure-function relationships of 4Aminobutyrate Aminotransferase, we analyzed 4Aminobutyrate Aminotransferase cDNA from pig brain. The inclusion bodies were formed when recombinant 4Aminobutyrate Aminotransferase was overexpressed in Escherichia coli. The unfolded overproduced proteins, were purified by hydroxylapatite chromatography in the presence of urea and refolded by a sequential dialysis method. The renatured protein regained its catalytic activity. The lysyl residue at the 330 position of the amino-acid sequence serves as the anchoring site of the cofactor pyridoxal 5′-P. To verify the catalytic site of 4Aminobutyrate Aminotransferase, lysine 330 was mutated to arginine by site-specific mutagenesis. Overexpression and purification of the mutated 4Aminobutyrate Aminotransferase (K330R) were performed by the same method used the purification of wild-type 4Aminobutyrate Aminotransferase. The purified and renatured K330R protein did not show the catalytic activity of wild type 4Aminobutyrate Aminotransferase. Furthermore, the mutated protein did not show any absorption band over the spectral range of 320-460 nm characteristic of pyridoxal 5′-P covalently linked to the protein. From the results presented here, it is concluded that lysine 330 is essential for the catalytic function of the Aminotransferase.

  • SCREENING AND SEQUENCE DETERMINATION OF A CDNA ENCODING THE HUMAN BRAIN 4Aminobutyrate Aminotransferase
    Gene, 1995
    Co-Authors: Yaa Difie Osei, Jorge E. Churchich

    Abstract:

    Abstract A human brain cDNA library constructed in the λZAP®II vector was screened using a fragment of pig brain cDNA encoding 4Aminobutyrate Aminotransferase (pGaba-t). A cDNA that encodes the human brain Gaba-t (hGaba-t) has been isolated from the library and sequenced. Using the GenBank and EMBL databases, comparison of the predicted amino-acid sequence of hGaba-t with the pig enzyme revealed 95.4% homology.

Anne M Evans – One of the best experts on this subject based on the ideXlab platform.

  • 2 pyrrolidinone and succinimide as clinical screening biomarkers for gaba transaminase deficiency anti seizure medications impact accurate diagnosis
    Frontiers in Neuroscience, 2019
    Co-Authors: Adam D Kennedy, Kirk L Pappan, Taraka R Donti, Mauricio R Delgado, Marwan Shinawi, Toni S Pearson, Seema R Lalani, William E Craigen, Reid V Sutton, Anne M Evans

    Abstract:

    : Broad-scale untargeted biochemical phenotyping is a technology that supplements widely accepted assays, such as organic acid, amino acid, and acylcarnitine analyses typically utilized for the diagnosis of inborn errors of metabolism. In this study, we investigate the analyte changes associated with 4Aminobutyrate Aminotransferase (ABAT, GABA transaminase) deficiency and treatments that affect GABA metabolism. GABA-transaminase deficiency is a rare neurodevelopmental and neurometabolic disorder caused by mutations in ABAT and resulting in accumulation of GABA in the cerebrospinal fluid (CSF). For that reason, measurement of GABA in CSF is currently the primary approach to diagnosis. GABA-transaminase deficiency results in severe developmental delay with intellectual disability, seizures, and movement disorder, and is often associated with death in childhood. Using an untargeted metabolomics platform, we analyzed EDTA plasma, urine, and CSF specimens from four individuals with GABA-transaminase deficiency to identify biomarkers by comparing the biochemical profile of individual patient samples to a pediatric-centric population cohort. Metabolomic analyses of over 1,000 clinical plasma samples revealed a rich source of biochemical information. Three out of four patients showed significantly elevated levels of the molecule 2-pyrrolidinone (Z-score ≥2) in plasma, and whole exome sequencing revealed variants of uncertain significance in ABAT. Additionally, these same patients also had elevated levels of succinimide in plasma, urine, and CSF and/or homocarnosine in urine and CSF. In the analysis of clinical EDTA plasma samples, the levels of succinimide and 2-pyrrolidinone showed a high level of correlation (R = 0.73), indicating impairment in GABA metabolism and further supporting the association with GABA-transaminase deficiency and the pathogenicity of the ABAT variants. Further analysis of metabolomic data across our patient population revealed the association of elevated levels of 2-pyrrolidinone with administration of vigabatrin, a commonly used anti-seizure medication and a known inhibitor of GABA-transaminase. These data indicate that anti-seizure medications may alter the biochemical and metabolomic data, potentially impacting the interpretation and diagnosis for the patient. Further, these data demonstrate the power of combining broad scale genotyping and phenotyping technologies to diagnose inherited neurometabolic disorders and support the use of metabolic phenotyping of plasma to screen for GABA-transaminase deficiency.

  • Data_Sheet_1_2-Pyrrolidinone and Succinimide as Clinical Screening Biomarkers for GABA-Transaminase Deficiency: Anti-seizure Medications Impact Accurate Diagnosis.xlsx
    , 2019
    Co-Authors: Adam D Kennedy, Kirk L Pappan, Taraka R Donti, Mauricio R Delgado, Marwan Shinawi, Toni S Pearson, Seema R Lalani, William E Craigen, Reid V Sutton, Anne M Evans

    Abstract:

    Broad-scale untargeted biochemical phenotyping is a technology that supplements widely accepted assays, such as organic acid, amino acid, and acylcarnitine analyses typically utilized for the diagnosis of inborn errors of metabolism. In this study, we investigate the analyte changes associated with 4Aminobutyrate Aminotransferase (ABAT, GABA transaminase) deficiency and treatments that affect GABA metabolism. GABA-transaminase deficiency is a rare neurodevelopmental and neurometabolic disorder caused by mutations in ABAT and resulting in accumulation of GABA in the cerebrospinal fluid (CSF). For that reason, measurement of GABA in CSF is currently the primary approach to diagnosis. GABA-transaminase deficiency results in severe developmental delay with intellectual disability, seizures, and movement disorder, and is often associated with death in childhood. Using an untargeted metabolomics platform, we analyzed EDTA plasma, urine, and CSF specimens from four individuals with GABA-transaminase deficiency to identify biomarkers by comparing the biochemical profile of individual patient samples to a pediatric-centric population cohort. Metabolomic analyses of over 1,000 clinical plasma samples revealed a rich source of biochemical information. Three out of four patients showed significantly elevated levels of the molecule 2-pyrrolidinone (Z-score ≥2) in plasma, and whole exome sequencing revealed variants of uncertain significance in ABAT. Additionally, these same patients also had elevated levels of succinimide in plasma, urine, and CSF and/or homocarnosine in urine and CSF. In the analysis of clinical EDTA plasma samples, the levels of succinimide and 2-pyrrolidinone showed a high level of correlation (R = 0.73), indicating impairment in GABA metabolism and further supporting the association with GABA-transaminase deficiency and the pathogenicity of the ABAT variants. Further analysis of metabolomic data across our patient population revealed the association of elevated levels of 2-pyrrolidinone with administration of vigabatrin, a commonly used anti-seizure medication and a known inhibitor of GABA-transaminase. These data indicate that anti-seizure medications may alter the biochemical and metabolomic data, potentially impacting the interpretation and diagnosis for the patient. Further, these data demonstrate the power of combining broad scale genotyping and phenotyping technologies to diagnose inherited neurometabolic disorders and support the use of metabolic phenotyping of plasma to screen for GABA-transaminase deficiency.

Cornelis Jakobs – One of the best experts on this subject based on the ideXlab platform.

  • A new case of GABA transaminase deficiency facilitated by proton MR spectroscopy
    Journal of Inherited Metabolic Disease, 2010
    Co-Authors: Megumi Tsuji, Noriko Aida, Takayuki Obata, Moyoko Tomiyasu, Noritaka Furuya, Kenji Kurosawa, Abdellatif Errami, K. Michael Gibson, Gajja S. Salomons, Cornelis Jakobs

    Abstract:

    Background Deficiency of 4Aminobutyrate Aminotransferase (GABA-T) is a rare disorder of GABA catabolism, with only a single sibship reported. We report on a third case, a Japanese female infant with severe psychomotor retardation and recurrent episodic lethargy with intractable seizures, with the diagnosis facilitated by proton magnetic resonance (MR) spectroscopy (^1H-MRS). Methods Neuroimaging was performed at the first episode of lethargy. For ^1H-MRS, locations were placed in the semioval center and the basal ganglia. Quantification of metabolite concentrations were derived using the LCModel. We confirmed the diagnosis subsequently by enzyme and molecular studies, which involved direct DNA sequence analysis and the development of a novel multiplex ligation-dependent probe amplification test. Results ^1H-MRS analysis revealed an elevated GABA concentration in the basal ganglia (2.9 mmol/l). Based on the results of quantitative ^1H-MRS and clinical findings, GABA-T deficiency was suspected and confirmed in cultured lymphoblasts. Molecular studies of the GABA-T gene revealed compound heterozygosity for a deletion of one exon and a missense mutation, 275G>A, which was not detected in 210 control chromosomes. Conclusions Our results suggest that excessive prenatal GABA exposure in the central nervous system (CNS) was responsible for the clinical manifestations of GABA transaminase deficiency. Our findings suggest the dual nature of GABA as an excitatory molecule early in life, followed by a functional switch to an inhibitory species later in development. Furthermore, quantitative ^1H-MRS appears to be a useful, noninvasive tool for detecting inborn errors of GABA metabolism in the CNS.

  • A new case of GABA transaminase deficiency facilitated by proton MR spectroscopy
    Journal of inherited metabolic disease, 2010
    Co-Authors: Megumi Tsuji, Noriko Aida, Takayuki Obata, Moyoko Tomiyasu, Noritaka Furuya, Kenji Kurosawa, Abdellatif Errami, K. Michael Gibson, Gajja S. Salomons, Cornelis Jakobs

    Abstract:

    Background
    Deficiency of 4Aminobutyrate Aminotransferase (GABA-T) is a rare disorder of GABA catabolism, with only a single sibship reported. We report on a third case, a Japanese female infant with severe psychomotor retardation and recurrent episodic lethargy with intractable seizures, with the diagnosis facilitated by proton magnetic resonance (MR) spectroscopy (1H-MRS).