Biochemical and Structural Properties of Mouse Kynurenine Aminotransferase III

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°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.     

Structural insight into the mechanism of substrate specificity of aedes kynurenine aminotransferase

Aedes aegypti kynurenine aminotransferase (AeKAT) is a multifunctional aminotransferase. It catalyzes the transamination of a number of amino acids and uses many biologically relevant alpha-keto acids as amino group acceptors. AeKAT also is a cysteine S-conjugate beta-lyase. The most important function of AeKAT is the biosynthesis of kynurenic acid, a natural antagonist of NMDA and alpha7-nicotinic acetylcholine receptors. Here, we report the crystal structures of AeKAT in complex with its best amino acid substrates, glutamine and cysteine. Glutamine is found in both subunits of the biological dimer, and cysteine is found in one of the two subunits. Both substrates form external aldemines with pyridoxal 5-phosphate in the structures. This is the first instance in which one pyridoxal 5-phosphate enzyme has been crystallized with cysteine or glutamine forming external aldimine complexes, cysteinyl aldimine and glutaminyl aldimine. All the units with substrate are in the closed conformation form, and the unit without substrate is in the open form, which suggests that the binding of substrate induces the conformation change of AeKAT. By comparing the active site residues of the AeKAT-cysteine structure with those of the human KAT I-phenylalanine structure, we determined that Tyr286 in AeKAT is changed to Phe278 in human KAT I, which may explain why AeKAT transaminates hydrophilic amino acids more efficiently than human KAT I does.     

Mitochondrial aspartate aminotransferase: a third kynurenate-producing enzyme in the mammalian brain

The tryptophan metabolite kynurenic acid (KYNA), which is produced enzymatically by the irreversible transamination of l-kynurenine, is an antagonist of alpha7 nicotinic and NMDA receptors and may thus modulate cholinergic and glutamatergic neurotransmission. Two kynurenine aminotransferases (KAT I and II) are currently considered the major biosynthetic enzymes of KYNA in the brain. In this study, we report the existence of a third enzyme displaying KAT activity in the mammalian brain. The novel KAT had a pH optimum of 8.0 and a low capacity to transaminate glutamine or alpha-aminoadipate (the classic substrates of KAT I and KAT II, respectively). The enzyme was inhibited by aspartate, glutamate, and quisqualate but was insensitive to blockade by glutamine or anti-KAT II antibodies. After purification to homogeneity, the protein was sequenced and the enzyme was identified as mitochondrial aspartate aminotransferase (mitAAT). Finally, the relative contributions of KAT I, KAT II, and mitAAT to total KAT activity were determined in mouse, rat, and human brain at physiological pH using anti-mitAAT antibodies. KAT II was most abundant in rat and human brain, while mitAAT played the major role in mouse brain. It remains to be seen if mitAAT participates in cerebral KYNA synthesis under physiological and/or pathological conditions in vivo.     

Decreased serum and red blood cell kynurenic acid levels in Alzheimer's disease

Kynurenine aminotransferases (KAT I and KAT II) are responsible for the transamination of kynurenine (KYN) to form kynurenic acid (KYNA), an excitatory amino acid receptor antagonist. Since these members of the kynurenine pathway (KP) are proposed to be involved in the pathogenesis of Alzheimer's dementia (AD), the activities of these enzymes and the levels of these metabolites were measured in the plasma and red blood cells (RBCs) of AD and control subjects together with the inheritance of the apolipoprotein (APOE) epsilon4 allele. KYNA levels were significantly decreased both in the plasma and in the RBCs in AD, but the levels of KYN and the activities of KAT I and KAT II remained unchanged. No association has been found with the possession of the epsilon4 allele. These findings indicate an altered peripheral KP in AD regardless of the APOE status of the probands.     

pH dependence, substrate specificity and inhibition of human kynurenine aminotransferase I.

Human kynurenine aminotransferase I/glutamine transaminase K (hKAT-I) is an important multifunctional enzyme. This study systematically studies the substrates of hKAT-I and reassesses the effects of pH, Tris, amino acids and alpha-keto acids on the activity of the enzyme. The experiments were comprised of functional expression of the hKAT-I in an insect cell/baculovirus expression system, purification of its recombinant protein, and functional characterization of the purified enzyme. This study demonstrates that hKAT-I can catalyze kynurenine to kynurenic acid under physiological pH conditions, indicates indo-3-pyruvate and cysteine as efficient inhibitors for hKAT-I, and also provides biochemical information about the substrate specificity and cosubstrate inhibition of the enzyme. hKAT-I is inhibited by Tris under physiological pH conditions, which explains why it has been concluded that the enzyme could not efficiently catalyze kynurenine transamination. Our findings provide a biochemical basis towards understanding the overall physiological role of hKAT-I in vivo and insight into controlling the levels of endogenous kynurenic acid through modulation of the enzyme in the human brain.     

Substrate specificity and structure of human aminoadipate aminotransferase/kynurenine aminotransferase II

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.     

Kynurenine Aminotransferase III and Glutamine Transaminase L Are Identical Enzymes that have Cysteine S-Conjugate β-Lyase Activity and Can Transaminate l-Selenomethionine

Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-l-selenocysteine (MSC) and l-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH₃SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH₃SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH₃SeH and/or seleno-keto acid metabolites.     

Alternative functions of the brain transsulfuration pathway represent an underappreciated aspect of brain redox biochemistry with significant potential for therapeutic engagement

Scientific appreciation for the subtlety of brain sulfur chemistry has lagged, despite understanding that the brain must maintain high glutathione (GSH) to protect against oxidative stress in tissue that has both a high rate of oxidative respiration and a high content of oxidation-prone polyunsaturated fatty acids. In fact, the brain was long thought to lack a complete transsulfuration pathway (TSP) for cysteine synthesis. It is now clear that not only does the brain possess a functional TSP, but brain TSP enzymes catalyze a rich array of alternative reactions that generate novel species including the gasotransmitter hydrogen sulfide (H₂S) and the atypical amino acid lanthionine (Lan). Moreover, TSP intermediates can be converted to unusual cyclic ketimines via transamination. Cell-penetrating derivatives of one such compound, lanthionine ketimine (LK), have potent antioxidant, neuroprotective, neurotrophic, and antineuroinflammatory actions and mitigate diverse neurodegenerative conditions in preclinical rodent models. This review will explore the source and function of alternative TSP products, and lanthionine-derived metabolites in particular. The known biological origins of lanthionine and its ketimine metabolite will be described in detail and placed in context with recent discoveries of a GSH- and LK-binding brain protein called LanCL1 that is proving essential for neuronal antioxidant defense; and a related LanCL2 homolog now implicated in immune sensing and cell fate determinations. The review will explore possible endogenous functions of lanthionine metabolites and will discuss the therapeutic potential of lanthionine ketimine derivatives for mitigating diverse neurological conditions including Alzheimer׳s disease, stroke, motor neuron disease, and glioma.     

Human kynurenine aminotransferase II--reactivity with substrates and inhibitors

Kynurenine aminotransferase (KAT) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the conversion of kynurenine, an intermediate of the tryptophan degradation pathway, into kynurenic acid, an endogenous antagonist of ionotropic excitatory amino acid receptors in the central nervous system. KATII is the prevalent isoform in mammalian brain and a drug target for the treatment of schizophrenia. We have carried out a spectroscopic and functional characterization of both the human wild-type KATII and a variant carrying the active site mutation Tyr142→Phe. The transamination and the β-lytic activity of KATII towards the substrates kynurenine and α-aminoadipate, the substrate analog β-chloroalanine and the inhibitors (R)-2-amino-4-(4-(ethylsulfonyl))-4-oxobutanoic acid and cysteine sulfinate were investigated with both conventional assays and a novel continuous spectrophotometric assay. Furthermore, for high-throughput KATII inhibitor screenings, an endpoint assay suitable for 96-well plates was also developed and tested. The availability of these assays and spectroscopic analyses demonstrated that (R)-2-amino-4-(4-(ethylsulfonyl))-4-oxobutanoic acid and cysteine sulfinate, reported to be KATII inhibitors, are poor substrates that undergo slow transamination.     

Sulfur-containing cyclic ketimines and imino acids. A novel family of endogenous products in the search for a role.

Aminoethylcysteine, lanthionine, cystathionine and cystine are mono-deaminated either by L-amino-acid oxidase or by a transaminase exhibiting the properties described for glutamine transaminase. The deaminated products cyclize producing the respective ketimines. Authentic samples of each ketimine were prepared by reacting the appropriate aminothiol compound with bromopyruvate, except cystine ketimine which required the interaction of thiopyruvate with cystine sulfoxide. Reduction of the first three mentioned ketimines with NaBH4 yields the respective derivatives with the saturated rings of thiomorpholine and hexahydrothiazepine. The same reduction is carried out enzymically by a reductase extracted from mammalian tissues. Properties of the members of this family of compounds are described. Gas chromatography followed by mass spectrometry permits the identification of most of these products. HPLC is very useful for the determination of the ketimines by taking advantage of specific absorbance at 380 nm obtained by prior derivatization with phenylisothiocyanate. Adaptation of these and other analytical procedures to biological samples disclosed the presence of most of these compounds in bovine brain and in human urine. By using [35S]lanthionine ketimine as a representative member of the ketimine group, the specific, high-affinity, saturable and reversible binding to bovine brain membranes has been demonstrated. The binding is removed by aminoethylcysteine ketimine and by cystathionine ketimine indicating the occurrence in bovine brain of a common binding site for ketimines. The reduced ketimines are totally ineffective in competing with [35S]lanthionine ketimine. Alltogether these findings are highly indicative for the existence in mammals of a novel class of endogenous sulfur-containing cyclic products provided with a possible neurochemical function to be investigated further.     

Bovine brain ketimine reductase

We report the purification from bovine brain of an NAD(P)H-dependent reductase which actively reduces a new class of cyclic unsaturated compounds, named ketimines. Ketimines arise from the transamination of some sulphur-containing amino acids, such as L-cystathionine, S-aminoethyl-L-cysteine and L-lanthionine. The enzyme also reduces delta 1-piperidine 2-carboxylate, the carbon analog of aminoethylcysteine ketimine. Some kinetic and molecular properties of this enzyme have been determined. Subcellular localization and regional brain distribution have also been studied. The ketimine reductase activity was found to be associated with the soluble fraction, and was located prevalently in the cerebellum and cerebral cortices. Cyclothionine and 1,4-thiomorpholine-3,5-dicarboxylic acid, the enzymatic reduction products of cystathionine ketimine and lanthionine ketimine, respectively, have been detected in bovine brain, thus suggesting a role of this enzyme in their biosynthesis.     

Transamination of L-cystathionine and related compounds by a bovine liver enzyme. Possible identification with glutamine transaminase

A transaminase which catalyses the monodeamination of L-cystathionine was purified 1100-fold with a yield of 15% from bovine liver. The monoketoderivative of cystathionine spontaneously produces the cyclic ketimine. Other sulfur-containing amino acids related to cystathionine such as cystine, lanthionine and aminoethylcysteine were also substrates for the enzyme. The relative molecular mass of the enzyme was determined to be 94 000 with a probable dimeric structure formed of identical subunits. The isoelectric point of the enzyme was at pH 5.0 and the maximal enzymatic activity was found at pH 9.0--9.2. Kinetic parameters for cystathionine and for the other sulfur amino acids as well as for some alpha-keto acids were also determined. Among the natural amino acids tested, glutamine, methionine and histidine were the best amino donors. The enzyme exhibited maximal activity toward phenylpyruvate and alpha-keto-gamma-methiolbutyrate as amino acceptors. The broad specificity of the enzyme leads us to infer that the cystathionine transaminase is very similar or identical to glutamine transaminase.     

Asparagine transaminase from rat liver.

Asparagine transaminase has been purified about 200-fold from rat liver. The enzyme has a broad specificity toward both amino acids and alpha-keto acids. Thus, amino acids substituted in the beta position such as asparagine, S-methylcysteine, phenylalanine, cysteine, serine, and aspartate are substrates. The enzyme is also active with alanine, methionine, homoserine, alpha-aminobutyrate, glutamine, and leucine. The enzyme has a high affinity for glyoxylate but the affinity falls off markedly through the series glyoxylate, pyruvate, alpha-ketoburyrate, alpha-Keto acids substituted in the beta or gamma position, such as alpha-ketosuccinamate, phenylpyruvate, p-hydroxyphenylpyruvate, alpha-keto-gamma-methiolburyrate, and alpha-keto-gamma-hydroxybutyrate, are substrates for the enzyme. Amino acids or alpha-keto acids possessing a branch point at the beta carbon are inactive. Kinetic analysis of the asparagine glyoxylate transamination reaction is consistent with a ping-pong mechanism.     

Characterisation and quantification of protein oxidative modifications and amino acid racemisation in powdered infant milk formula

Modification of proteins in infant milk formula (IF) is of major concern to the dairy industry and consumers. Thermal treatment is required for microbiological safety, but heat, light, metal-ions and other factors may induce oxidative damage, and be a health risk. In this study protein modifications in IFs were quantified. IFs contained both reducible (disulphide) and non-reducible (di-tyrosine, lanthionine, lysinoalanine) protein cross-links. Dehydroalanine and the cross-linked species lanthionine and lysinoalanine were detected. Protein carbonyls were detected predominantly on high molecular mass materials. Oxidation products of phenylalanine (m-tyrosine), tryptophan (N-formylkynurenine, kynurenine, 3-hydroxykynurenine), tyrosine (di-tyrosine) and methionine (methionine sulphoxide) were detected, consistent with amino acid modification. Higher levels of most of the markers of protein modification were present in the hydrolysed protein brand, when compared to the conventional IF samples, indicative of increased damage during additional processing. Significant levels of racemised (D-) amino acids were present. These data indicate that amino acids in proteins in IFs are modified to a significant extent during manufacture, with hydrolysed IF being particularly prone.     

Role of mitochondrial transamination in branched chain amino acid metabolism

Oxidative decarboxylation and transamination of 1-14C-branched chain amino and alpha-keto acids were examined in mitochondria isolated from rat heart. Transamination was inhibited by aminooxyacetate, but not by L-cycloserine. At equimolar concentrations of alpha-ketoiso[1-14C]valerate (KIV) and isoleucine, transamination was increased by disrupting the mitochondria with detergent which suggests transport may be one factor affecting the rate of transamination. Next, the subcellular distribution of the aminotransferase(s) was determined. Branched chain aminotransferase activity was measured using two concentrations of isoleucine as amino donor and [1-14C]KIV as amino acceptor. The data show that branched chain aminotransferase activity is located exclusively in the mitochondria in rat heart. Metabolism of extramitochondrial branched chain alpha-keto acids was examined using 20 microM [1-14C]KIV and alpha-ketoiso[1-14C]caproate (KIC). There was rapid uptake and oxidation of labeled branched chain alpha-keto acid, and, regardless of the experimental condition, greater than 90% of the labeled keto acid substrate was metabolized during the 20-min incubation. When a branched chain amino acid (200 microM) or glutamate (5 mM) was present, 30-40% of the labeled keto acid was transaminated while the remainder was oxidized. Provision of an alternate amino acceptor in the form of alpha-keto-glutarate (0.5 mM) decreased transamination of the labeled KIV or KIC and increased oxidation. Metabolism of intramitochondrially generated branched chain alpha-keto acids was studied using [1-14C]leucine and [1-14C]valine. Essentially all of the labeled branched chain alpha-keto acid produced by transamination of [1-14C]leucine or [1-14C]valine with a low concentration of unlabeled branched chain alpha-keto acid (20 microM) was oxidized. Further addition of alpha-ketoglutarate resulted in a significant increase in the rate of labeled leucine or valine transamination, but again most of the labeled keto acid product was oxidized. Thus, catabolism of branched chain amino acids will be favored by a high concentration of mitochondrial alpha-ketoglutarate and low intramitochondrial glutamate.     

Perinatal kynurenine pathway metabolism in the normal and asphyctic rat brain

Summary. The kynurenine pathway of tryptophan degradation contains several metabolites which may influence brain physiology and pathophysiology. The brain content of one of these compounds, kynurenic acid (KYNA), decreases precipitously around the time of birth, possibly to avoid deleterious N-methyl-D-aspartate (NMDA) receptor blockade during the perinatal period. The present study was designed to determine the levels of KYNA, the free radical generator 3-hydroxykynurenine (3-HK), and their common precursor L-kynurenine (L-KYN) between gestational day 16 and adulthood in rat brain and liver. The cerebral activities of the biosynthetic enzymes of KYNA and 3-HK, kynurenine aminotransferases (KATs) I and II and kynurenine 3-hydroxylase, respectively, were measured at the same ages. Additional studies were performed to assess whether and to what extent kynurenines in the immature brain derive from the mother, and to examine the short-term effects of birth asphyxia on brain KYNA and 3-HK levels. The results revealed that 1) the brain and liver content of L-KYN, KYNA and 3-HK is far higher pre-term than postnatally; 2) KAT I and kynurenine 3-hydroxylase activities are quite uniform between E-16 and adulthood, whereas KAT II activity rises sharply after postnatal day 14; 3) during the perinatal period, KYNA, but not L-KYN, may originate in part from the maternal circulation; and 4) oxygen deprivation at birth affects the brain content of both KYNA and 3-HK 1 h but not 24 h later. Received August 31, 1999 Accepted September 20, 1999     

Purification and Characterization of Kynurenine Aminotransferase I from Human Brain

Abstract: Two kynurenine aminotransferases (KATs), arbitrarily termed KAT I and KAT II, are capable of producing the neuroinhibitory brain metabolite kynurenic acid from l -kynurenine in human brain tissue. Here we describe the purification of KAT I to homogeneity and the subsequent characterization of the enzyme using physicochemical, biochemical, and immunological methods. KAT I was purified from human brain ∼2,000-fold with a yield of 2%. Assessed by polyacrylamide gel electrophoresis, KAT I migrated toward the anode as a single protein with a mobility of 0.5. The pure enzyme was found to be a dimer consisting of two identical subunits of ∼60 kDa. Among several oxo acids tested, KAT I showed highest activity with 2-oxoisocaproate. Kinetic analyses of the pure enzyme revealed an absolute K _m of 2.0 m M and 10.0 m M for l -kynurenine and pyruvate, respectively. KAT I activity was substantially inhibited by l -glutamine, l -phenylalanine, and l -tryptophan, using either pyruvate (1 m M ) or 2-oxoisocaproate (1 m M ) as a cosubstrate. l -Tryptophan inhibited enzyme activity noncompetitively with regard to pyruvate ( K _i = 480 µ M ) and competitively with regard to l -kynurenine ( K _i = 200 µ M ). Anti-KAT I antibodies were produced against pure KAT I and were partially purified by conventional techniques. Immunotitration and immunoblotting analyses confirmed that KAT I is clearly distinct from both human KAT II and rat kynurenine-pyruvate aminotransferase. Pure human KAT I and its antibody will serve as valuable tools in future studies of kynurenic acid production in the human brain under physiological and pathological conditions.     

Enzymatic oxidation of L-homocysteine

Homocyst(e)ine, a normal metabolite, accumulates in certain inborn errors of sulfur amino acid metabolism. Since many amino acids are converted by enzymatic oxidation and by transamination to the corresponding alpha-keto acid analogs and related products, which may exert inhibitory effects on metabolism, and because the alpha-keto acid analog of homocysteine has not yet been prepared, the enzymatic oxidation of homocysteine was investigated with the aim of obtaining alpha-keto-gamma-mercaptobutyric acid. Oxidation of DL-homocysteine by L-amino acid oxidase led to formation of at least seven products that react with 2,4-dinitrophenylhydrazine; of these, five were identified: alpha-keto-gamma-mercaptobutyrate, the mono and diketo analogs of homolanthionine, and the mono and diketo analogs of homocystine. In addition, one product was tentatively identified as alpha-ketomercaptobutyric acid gamma-thiolactone. In the course of this work alpha-keto-gamma-mercaptobutyrate was found to be a substrate of lactate dehydrogenase. L-Homocysteine and its alpha-keto acid analog were shown to be substrates of glutamate dehydrogenase and kidney glutamine transaminase. DL-Homocysteine reacts readily with alpha-keto acids to form stable hemithioketals, which were found to be substrates of L- and D-amino acid oxidases. A scheme is presented which integrates some of the complexities involved in the oxidation metabolism of homocyst(e)ine. The significance of these findings is considered in relation to the toxicity of homocysteine, which accumulates in certain pathological states.