The Synthesis of Kynurenic Acid in Mammals: An Updated Kynurenine Aminotransferase Structural KATalogue

Abstract

Kynurenic acid (KYNA) is a bioactive compound that is produced along the kynurenine pathway (KP) during tryptophan degradation. In a few decades, KYNA shifted from being regarded a poorly characterized by-product of the KP to being considered a main player in many aspects of mammalian physiology, including the control of glutamatergic and cholinergic synaptic transmission, and the coordination of immunomodulation. The renewed attention being paid to the study of KYNA homeostasis is justified by the discovery of selective and potent inhibitors of kynurenine aminotransferase II, which is considered the main enzyme responsible for KYNA synthesis in the mammalian brain. Since abnormally high KYNA levels in the central nervous system have been associated with schizophrenia and cognitive impairment, these inhibitors promise the development of novel anti-psychotic and pro-cognitive drugs. Here, we summarize the currently available structural information on human and rodent kynurenine aminotransferases (KATs) as the result of global efforts aimed at describing the full complement of mammalian isozymes. These studies highlight peculiar features of KATs that can be exploited for the development of isozyme-specific inhibitors. Together with the optimization of biochemical assays to measure individual KAT activities in complex samples, this wealth of knowledge will continue to foster the identification and rational design of brain penetrant small molecules to attenuate KYNA synthesis, i.e., molecules capable of lowering KYNA levels without exposing the brain to the harmful withdrawal of KYNA-dependent neuroprotective actions.

Keywords: PLP enzyme; crystal structure; kynurenic acid; kynurenine aminotransferase; kynurenine pathway.

Figure 1

The kynurenine pathway with a focus on KYNA synthesis. (A) NFK, N-formylkynurenine; L-KYN, L-kynurenine; KYNA, kynurenic acid; 3-HK, 3-hydroxykynurenine; XA, xanthurenic acid; AA, anthranilic acid; 3-HAA, 3-hydroxyanthranilic acid; ACMS, 2-amino-3-carboxymuconate-6-semialdehyde; AMS, 2-aminomuconate-6-semialdehyde; PA, picolinic acid; CBA, cinnabarinic acid; QA, quinolinic acid; NAD, nicotinamide adenine dinucleotide. Inset: 1. At the beginning of the transamination reaction, the catalytic lysine of KAT is bound to the PLP molecule. 2. The α-amino group of L-KYN substitutes for Lys and binds to the cofactor, forming an external aldimine intermediate. 3. At the end of the first half-reaction, the resulting α-keto acid undergoes an intramolecular condensation reaction, releasing the final product, KYNA, and leaving the cofactor in the pyridoxamine phosphate (PMP) form, then, an α-keto acid is required to bring the enzyme back to its initial state through a series of equivalent reversed reactions (not shown). (B) The multiplicity of KYNA actions inside and outside the CNS. KYNA acts as an antagonist of specific receptors both at the synapses and at extra-synaptic sites (only selected targets are shown); KYNA possesses immunomodulatory properties mainly by behaving as an agonist/ligand of GPR35 and AHR. KYNA participates in the direct scavenging of ROS; BBB, blood-brain barrier. (C) Analysis of protein sequence similarity among different KATs, including representatives of non-mammalian KATs (Mj, Methanocaldococcus jannaschii; Ph, Pyrococcus horikoshii; Ae, Aedes aegypti; Ag, Anopheles gambiae; HKT, 3-hydroxykynurenine aminotransferase). The values in brackets refer to the percentage sequence identity to the corresponding human isoenzyme; ^*no significant sequence identity. The phylogenetic tree was generated by iTOL (Letunic and Bork, 2016). (D) Comparison of selected kinetic constants for the transformation of KYN to KYNA and 3-HK to XA catalyzed by the indicated KATs; (Han et al., 2004, 2008a, 2009a, 2010a).

Figure 2

Structural features and properties of mammalian KATs. (A) In each KAT dimer, the “A” subunit appears in color, the “B” subunit appears in gray, and the red star labels one of the two identical active sites, the dotted circles frame the peculiar structural features of hKAT II. (B) Close-ups of the catalytic cavity of hKAT I in different ligand-bound states. (C) The active site of mKAT III in complex with HEPES, which adopts two alternative conformations. (D) Zoomed views of the hKAT II active site in complex with two irreversible inhibitors (1 and 2). In each image, the protein backbone is depicted as a cartoon, the selected residue side chain is depicted as a ball-and-stick model, the asterisk labels residues belonging to one subunit of the dimer, and the arrows indicate the major rearrangements discussed in the text. The PDB codes appear in brackets. As a matter of clarity, the images, which correspond to optimally superimposed structures, are presented side by side. The figures have been generated by PyMol (www.pymol.org).