Crystal Structure of Human Kynurenine Aminotransferase I

Rossi, Franca; Han, Qian; Li, Junsuo; Li, Jianyong; Rizzi, Menico

doi:10.1074/jbc.m409291200

Cited by 73 publications

(87 citation statements)

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“…Although many studies have dealt with the biochemical characteristics of mammalian KAT I and KAT II (3,4,9,16,19,25,49), little is known about the biochemical activity of KAT III. Likewise, the crystal structures of human KAT I (hKAT I) (48) and its homologues, glutamine-phenylpyruvate aminotransferase from Thermus thermophilus HB8 (13) and KAT from a mosquito, Aedes aegypti (AeKAT) (22), have been determined, as well as the crystal structure of hKAT II (26,47) and its homologue from Pyrococcus horikoshii (7). Although KAT III has been cloned from mice, rats, and humans as a member of the mammalian KAT family (60), its structure has not been determined for any of these species.…”

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Biochemical and Structural Properties of Mouse Kynurenine Aminotransferase III

Han

Robinson

Cai

et al. 2009

Molecular and Cellular Biology

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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.

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confidence: 99%

Biochemical and Structural Properties of Mouse Kynurenine Aminotransferase III

Han

Robinson

Cai

et al. 2009

Molecular and Cellular Biology

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112

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“…However, in our hands, 4-(2-aminophenyl)-4-oxobutyric acid does not inhibit the human KAT I isozyme in the millimolar range, providing a proof of principle that selective inhibitors toward the mosquito enzyme can be identified. We previously reported the 3D structure of human KAT I in complex with the substrate analogue L-Phe, identifying the structural determinants responsible for ligand binding and catalysis (20). Human KAT I and Ag-HKT belong to different subfamilies of PLP-dependent aminotransferases, show only 14% sequence identity, and can be superposed with a rms deviation of 5.0 Å for 320 C␣ pairs.…”

Section: Resultsmentioning

confidence: 99%

“…In each monomer, three distinct regions could be defined: an extended N-terminal arm, a large N-terminal domain, and a small C-terminal domain. The Ag-HKT N-terminal large domain is preceded by a random coiled stretch (residues 3-7 and 11-18), interrupted by an ␣-helical turn (residues 8-10) and ending in a short ␤-strand (residues [19][20][21]. This arm runs parallel to the surface of the large domain of the opposite monomer, between the ␣-helices H3 and H12, where it comes in close contact with the solvent-exposed side chains of several residues, thus contributing to the stability of the functional dimer (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The biochemical description of the kynurenine pathway from a number of vertebrates revealed that 3-HK can undergo two alternative fates: either (i) it is hydrolyzed by a specific kynureninase to alanine and 3-hydroxyanthranilic acid, feeding the de novo synthesis of the essential cofactor NAD (17), or (ii) it can be directly converted into the more chemically stable compound xanthurenic acid (XA) by irreversible transamination. In rodents and humans, this latter function is provided by the same kynurenine aminotransferase (KAT) isozymes that are responsible for the pyridoxal 5Ј-phosphate (PLP)-dependent transformation of L-KYN into the neuroprotective and anticonvulsant NMDA receptor antagonist kynurenic acid (KA) (18)(19)(20). In Anopheles gambiae, the vector of the most deadly malaria parasite Plasmodium falciparum, 3-HK hydrolytic activity has never been documented, even though 3-HK-dependent XA synthesis might represent the only means to prevent the accumulation of this toxic molecule.…”

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Crystal structure of the Anopheles gambiae 3-hydroxykynurenine transaminase

Rossi

Garavaglia²,

Giovenzana³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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In Anopheles gambiae, the vector for the most deadly malaria parasite Plasmodium falciparum, xanthurenic acid (XA) plays a key role in parasite gametogenesis and fertility. In mosquitoes, XA is produced by transamination of 3-hydroxykynurenine (3-HK), a reaction that represents the main route to prevent the accumulation of the potentially toxic 3-HK excess. Interfering with XA metabolism in A. gambiae therefore appears an attractive avenue for the development of malaria transmission-blocking drugs and insecticides. We have determined the crystal structure of A. gambiae 3-HK transaminase in its pyridoxal 5 -phosphate form and in complex with a newly synthesized competitive enzyme inhibitor. Structural inspection of the enzyme active site reveals the key molecular determinants for ligand recognition and catalysis. Major contributions toward inhibitor binding are provided by a salt bridge between the inhibitor carboxylate and Arg-356 and by a remarkable hydrogen bond network involving the anthranilic moiety of the inhibitor and backbone atoms of residues Gly-25 and Asn-44. This study may be useful for the structurebased design of specific enzyme inhibitors of potential interest as antimalarial agents.kynurenine pathway ͉ xanthurenic acid

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“…Interestingly, cysteine has an intriguing effect on AeKAT activity toward kynurenine, enhancing its activity at relatively low concentrations and inhibiting its activity at higher concentrations (5,18). Crystal structures of AeKAT, glutamine:phenylpyruvate aminotransferase from T. thermophilus, and human KAT I have been available for substrate recognition study (19)(20)(21). They are fold type I aminotransferases (22,23), characterized by the presence of an N-terminal arm, a small domain, and a large domain.…”

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Structural Insight into the Mechanism of Substrate Specificity of Aedes Kynurenine Aminotransferase^,

et al. 2008

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Aedes aegypti kynurenine aminotransferase (AeKAT) is a multifunctional aminotransferase. It catalyzes the transamination of a number of amino acids and uses many biologically relevant α-keto acids as amino group acceptors. AeKAT also is a cysteine S-conjugate β-lyase. The most important function of AeKAT is the biosynthesis of kynurenic acid, a natural antagonist of NMDA and α7-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.The sequence of Aedes aegypti kynurenine aminotransferase (AeKAT) 1 is 47% identical with that of human KAT I and 51.9% identical with that of human KAT III (1). Human, rat, and mouse KAT I enzymes have been extensively studied (2,3). Mammalian KAT I, also called glutamine transaminase K (GTK), is a multifunctional aminotransferase that acts on a number of amino acids (glutamine, cysteine, phenylalanine, kynurenine, methionine, etc.) and uses many biologically relevant keto acids as amino group acceptors (3,4). AeKAT and a glutamine:phenylpyruvate aminotransferase from Thermus thermophilus, homologues of mammalian KAT I, have also been systematically characterized (5,6). One of the enzymatic products of KAT I is kynurenic acid (KYNA), which is the only known endogenous antagonist of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors (7-9). KYNA is also the antagonist of the α7-nicotinic acetylcholine receptor (10-12). Very † This work was supported by Grant AI 44399 from the National Institutes of Health. ‡ The atomic coordinates and structure factors (entries 2r5c and 2r5e) have been deposited in the Protein Data Bank. Many other functions have been proposed for mammalian KAT I enzymes, including maintaining low levels of phenylpyruvate, closing the methionine salvage pathway, regulating α-keto acid levels, sparing the essential amino acids, and maintaining a continual equilibrium among the amino acids (3,17). However, quantitatively, the most important amine donor for KAT I enzymes in vivo is glutamine. The product of glutamine transamination (α-ketoglutaramate) is rapidly remov...

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Crystal Structure of Human Kynurenine Aminotransferase I

Cited by 73 publications

References 51 publications

Biochemical and Structural Properties of Mouse Kynurenine Aminotransferase III

Biochemical and Structural Properties of Mouse Kynurenine Aminotransferase III

Crystal structure of the Anopheles gambiae 3-hydroxykynurenine transaminase

Structural Insight into the Mechanism of Substrate Specificity of Aedes Kynurenine Aminotransferase^,

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Crystal Structure of Human Kynurenine Aminotransferase I

Cited by 73 publications

References 51 publications

Biochemical and Structural Properties of Mouse Kynurenine Aminotransferase III

Biochemical and Structural Properties of Mouse Kynurenine Aminotransferase III

Crystal structure of the Anopheles gambiae 3-hydroxykynurenine transaminase

Structural Insight into the Mechanism of Substrate Specificity of Aedes Kynurenine Aminotransferase,

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Structural Insight into the Mechanism of Substrate Specificity of Aedes Kynurenine Aminotransferase^,