Purification and properties of kynurenine aminotransferase from rat kidney.
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ABSTRACT:
SUBMITTER: Mawal MR
PROVIDER: S-EPMC1151645 | biostudies-other | 1991 Oct
REPOSITORIES: biostudies-other
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Project description:Kynurenine aminotransferase III (KAT III) has been considered to be involved in the production of mammalian brain kynurenic acid (KYNA), which plays an important role in protecting neurons from overstimulation by excitatory neurotransmitters. The enzyme was identified based on its high sequence identity with mammalian KAT I, but its activity toward kynurenine and its structural characteristics have not been established. In this study, the biochemical and structural properties of mouse KAT III (mKAT III) were determined. Specifically, mKAT III cDNA was amplified from a mouse brain cDNA library, and its recombinant protein was expressed in an insect cell protein expression system. We established that mKAT III is able to efficiently catalyze the transamination of kynurenine to KYNA and has optimum activity at relatively basic conditions of around pH 9.0 and at relatively high temperatures of 50 to 60 degrees C. In addition, mKAT III is active toward a number of other amino acids. Its activity toward kynurenine is significantly decreased in the presence of methionine, histidine, glutamine, leucine, cysteine, and 3-hydroxykynurenine. Through macromolecular crystallography, we determined the mKAT III crystal structure and its structures in complex with kynurenine and glutamine. Structural analysis revealed the overall architecture of mKAT III and its cofactor binding site and active center residues. This is the first report concerning the biochemical characteristics and crystal structures of KAT III enzymes and provides a basis toward understanding the overall physiological role of mammalian KAT III in vivo and insight into regulating the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.
| S-EPMC2630683 | biostudies-literature
Project description:Kynurenine aminotransferase isozymes (KATs 1-4) are members of the pyridoxal-5'-phosphate (PLP)-dependent enzyme family, which catalyse the permanent conversion of l-kynurenine (l-KYN) to kynurenic acid (KYNA), a known neuroactive agent. As KATs are found in the mammalian brain and have key roles in the kynurenine pathway, involved in different categories of central nervous system (CNS) diseases, the KATs are prominent targets in the quest to treat neurodegenerative and cognitive impairment disorders. Recent studies suggest that inhibiting these enzymes would produce effects beneficial to patients with these conditions, as abnormally high levels of KYNA are observed. KAT-1 and KAT-3 share the highest sequence similarity of the isozymes in this family, and their active site pockets are also similar. Importantly, KAT-2 has the major role of kynurenic acid production (70%) in the human brain, and it is considered therefore that suitable inhibition of this isozyme would be most effective in managing major aspects of CNS diseases. Human KAT-2 inhibitors have been developed, but the most potent of them, chosen for further investigations, did not proceed in clinical studies due to the cross toxicity caused by their irreversible interaction with PLP, the required cofactor of the KAT isozymes, and any other PLP-dependent enzymes. As a consequence of the possibility of extensive undesirable adverse effects, it is also important to pursue KAT inhibitors that reversibly inhibit KATs and to include a strategy that seeks compounds likely to achieve substantial interaction with regions of the active site other than the PLP. The main purpose of this treatise is to review the recent developments with the inhibitors of KAT isozymes. This treatise also includes analyses of their crystallographic structures in complex with this enzyme family, which provides further insight for researchers in this and related studies.
| S-EPMC4926479 | biostudies-literature
Project description:Previous studies with rat kidney preparations indicated that 2-aminoadipate aminotransferase (AadAT) and kynurenine aminotransferase (KAT) activities are properties of a single protein. We found that bovine kidney contains an appreciable amount of AadAT activity, but lacks KAT activity. AadAT from bovine and rat kidney extracts were purified to electrophoretic homogeneity. The purification procedure included fractionation with (NH1)2SO1, heat treatment, DEAE-cellulose chromatography and hydroxyapatite chromatography. Physical and kinetic properties, such as pH optima, Km for substrates, Mr, electrophoretic mobility and inhibition by dicarboxylic acids of bovine kidney AadAT, were similar to those of the rat kidney enzyme. However, bovine kidney AadAT differed from rat kidney AadAT in substrate specificity, amino acid composition and stability when stored. The titration curve of bovine kidney AadAT was also different from that of the rat kidney enzyme. The results suggest that bovine kidney AadAT may have some structural similarity to rat kidney AadAT and that the structural differences observed between the two enzymes may explain the absence of KAT activity in bovine kidney.
| S-EPMC1138897 | biostudies-other
Project description:KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to alpha-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested alpha-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with alpha-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15-33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.
| S-EPMC2559858 | biostudies-literature
Project description:Pyruvate (glyoxylate) aminotransferase from rat liver peroxisomes was highly purified and characterized. The enzyme preparation has a mol.wt. of approx. 80,000 with two identical subunits, and isoelectric point of 8.0 and a pH optimum between 8.0 and 8.5. The enzyme catalysed transamination between a number of L-amino acids and pyruvate or glyoxylate. The effective amino acceptors were pyruvate, phenylpyruvate and glyoxylate with serine, and glyoxylate and phenylpyruvate with alanine as amino donor. These properties and kinetic parameters of the enzyme are remarkably similar to those previously described for mitochondrial alanine-glyoxylate aminotransferase isoenzyme 1 from glucagon-injected rat liver [Noguchi, Okuno, Takada, Minatogawa, Okai & Kido (1978, Biochem. J. 169, 113-122].
| S-EPMC1186129 | biostudies-other
Project description:l-Arginase from rat kidney was partially purified and some properties were compared with those of l-arginase of rat liver. The kidney enzyme was firmly bound to the mitochondrial fraction and after solubilization required arginine or an unknown factor in tissue extracts for stabilization after dialysis. The two enzymes differed also in stability with respect to acetone treatment, heating or freezing. In further contrast with liver arginase, arginase from kidney was not adsorbed to CM-cellulose at pH7.5 and its activity was not increased by incubation with Mn(2+). Other differences were seen in relative specificities for substrates, ratio of hydrolysis rates with high and low concentrations of arginine and effects of certain inhibitors. Antisera prepared to pure liver arginase did not cross-react with partially purified kidney arginase.
| S-EPMC1177768 | biostudies-other
Project description:Kynurenine aminotransferase from Pyrococcus horikoshii OT3 (PhKAT), which is a homodimeric protein, catalyzes the conversion of kynurenine (KYN) to kynurenic acid (KYNA). We analyzed the transaminase reaction mechanisms of this protein with pyridoxal-5'-phosphate (PLP), KYN and ?-ketoglutaric acid (2OG) or oxaloacetic acid (OXA). 2OG significantly inhibited KAT activities in kinetic analyses, suggesting that a KYNA biosynthesis is allosterically regulated by 2OG. Its inhibitions evidently were unlocked by KYN. 2OG and KYN functioned as an inhibitor and activator in response to changes in the concentrations of KYN and 2OG, respectively. The affinities of one subunit for PLP or 2OG were different from that of the other subunit, as confirmed by spectrophotometry and isothermal titration calorimetry, suggesting that the difference of affinities between subunits might play a role in regulations of the KAT reaction. Moreover, we identified two active and allosteric sites in the crystal structure of PhKAT-2OG complexes. The crystal structure of PhKAT in complex with four 2OGs demonstrates that two 2OGs in allosteric sites are effector molecules which inhibit the KYNA productions. Thus, the combined data lead to the conclusion that PhKAT probably is regulated by allosteric control machineries, with 2OG as the allosteric inhibitor.
| S-EPMC3391261 | biostudies-literature
Project description:Mammalian mAspAT (mitochondrial aspartate aminotransferase) is recently reported to have KAT (kynurenine aminotransferase) activity and plays a role in the biosynthesis of KYNA (kynurenic acid) in rat, mouse and human brains. This study concerns the biochemical and structural characterization of mouse mAspAT. In this study, mouse mAspAT cDNA was amplified from mouse brain first stand cDNA and its recombinant protein was expressed in an Escherichia coli expression system. Sixteen oxo acids were tested for the co-substrate specificity of mouse mAspAT and 14 of them were shown to be capable of serving as co-substrates for the enzyme. Structural analysis of mAspAT by macromolecular crystallography revealed that the cofactor-binding residues of mAspAT are similar to those of other KATs. The substrate-binding residues of mAspAT are slightly different from those of other KATs. Our results provide a biochemical and structural basis towards understanding the overall physiological role of mAspAT in vivo and insight into controlling the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.
| S-EPMC3046307 | biostudies-literature
Project description:Kynurenic acid, a metabolite of the kynurenine pathway of tryptophan degradation, acts as an endogenous antagonist of alpha7 nicotinic and NMDA receptors and is implicated in a number of neurophysiological and neuropathological processes including cognition and neurodegenerative events. Therefore, kynurenine aminotransferase II (KAT II/AADAT), the enzyme responsible for the formation of the majority of neuroactive kynurenic acid in the brain, has prompted significant interest. Using immunohistochemistry, this enzyme was localized primarily in astrocytes throughout the adult rat brain, but detailed neuroanatomical studies are lacking. Here, we employed quantitative in situ hybridization to analyze the relative expression of KAT II mRNA in the brain of rats under normal conditions and 6 h after the administration of lipopolysaccharides (LPSs). Specific hybridization signals for KAT II were detected, with the highest expression in the subventricular zone (SVZ), the rostral migratory stream and the floor of the third ventricle followed by the corpus callosum and the hippocampus. This pattern of mRNA expression was paralleled by differential protein expression, determined by serial dilutions of antibodies (up to 1:1 million), and was confirmed to be primarily astrocytic in nature. The mRNA signal in the SVZ and the hippocampus was substantially increased by the LPS treatment without detectable changes elsewhere. These results demonstrate that KAT II is expressed in the rat brain in a region-specific manner and that gene expression is sensitive to inflammatory processes. This suggests an unrecognized role for kynurenic acid in the brain's germinal zones.
| S-EPMC5766363 | biostudies-literature
Project description:BackgroundKynurenine aminotransferase 3 (KAT3) catalyzes the transamination of Kynurenine to kynurenic acid, and is identical to cysteine conjugate beta-lyase 2 (CCBL2) and glutamine transaminase L (GTL). GTL was previously purified from the rat liver and considered as a liver type glutamine transaminase. However, because of the substrate overlap and high sequence similarity of KAT3 and KAT1, it was difficult to assay the specific activity of each KAT and to study the enzyme localization in animals.MethodsKAT3 transcript and protein levels as well as enzyme activity in the liver and kidney were analyzed by regular reverse transcription-polymerase chain reaction (RT-PCR), real time RT-PCR, biochemical activity assays combined with a specific inhibition assay, and western blotting using a purified and a highly specific antibody, respectively.ResultsThis study concerns the comparative biochemical characterization and localization of KAT 3 in the mouse. The results showed that KAT3 was present in both liver and kidney of the mouse, but was much more abundant in the kidney than in the liver. The mouse KAT3 is more efficient in transamination of glutamine with indo-3-pyruvate or oxaloacetate as amino group acceptor than the mouse KAT1.ConclusionsMouse KAT3 is a major glutamine transaminase in the kidney although it was named a liver type transaminase.General significanceOur data highlights KAT3 as a key enzyme for studying the nephrotoxic mechanism of some xenobiotics and the formation of chemopreventive compounds in the mouse kidney. This suggests tissue localizations of KAT3/GTL/CCBL2 in other animals may be carefully checked.
| S-EPMC5613967 | biostudies-literature