High resolution crystal structures of human kynurenine aminotransferase-I bound to PLP cofactor, and in complex with aminooxyacetate.

In this study, we report two high-resolution structures of the pyridoxal 5' phosphate (PLP)-dependent enzyme kynurenine aminotransferase-I (KAT-I). One is the native structure with the cofactor in the PLP form bound to Lys247 with the highest resolution yet available for KAT-I at 1.28 Å resolution, and the other with the general PLP-dependent aminotransferase inhibitor, aminooxyacetate (AOAA) covalently bound to the cofactor at 1.54 Å. Only small conformational differences are observed in the vicinity of the aldimine (oxime) linkage with which the PLP forms the Schiff base with Lys247 in the 1.28 Å resolution native structure, in comparison to other native PLP-bound structures. We also report the inhibition of KAT-1 by AOAA and aminooxy-phenylpropionic acid (AOPP), with IC50s of 13.1 and 5.7 µM, respectively. The crystal structure of the enzyme in complex with the inhibitor AOAA revealed that the cofactor is the PLP form with the external aldimine linkage. The location of this oxime with the PLP, which forms in place of the native internal aldimine linkage of PLP of the native KAT-I, is away from the position of the native internal aldimine, with the free Lys247 substantially retaining the orientation of the native structure. Tyr101, at the active site, was observed in two conformations in both structures.

Expression, purification and crystallization of human kynurenine aminotransferase 2 exploiting a highly optimized codon set.

Kynurenine aminotransferase (KAT) is a pyridoxal-5'-phosphate (PLP) dependent enzyme that catalyses kynurenine (KYN) to kynurenic acid (KYNA), a neuroactive product in the tryptophan metabolic pathway. Evidence suggests that abnormal levels of KYNA are involved in many neurodegenerative diseases such as Parkinson's disease, Huntington's disease, Alzheimer's disease and schizophrenia. Reducing KYNA production through inhibiting kynurenine aminotransferase 2 (KAT2) would be a promising approach to understanding and treating the related neurological and mental disorders. In this study we used an optimized codon sequence to overexpress histidine-tagged human KAT2 (hKAT2) using an Escherichia coli expression system. After a single step of Ni-NTA based purification the purified protein (>95%) was confirmed to be active by an HPLC based activity assay and was crystallized using the hanging-drop vapour diffusion method. The crystal system represents a novel space group, and a complete X-ray diffraction data set was collected to 1.83 Å resolution, and higher resolution data than for any reported native human KAT2 structure. The optimised method of protein production provides a fast and reliable technique to generate large quantities of active human KAT2 suitable for future small-molecule lead compound screening and structural design work.

Kynurenine Aminotransferases and the Prospects of Inhibitors for the Treatment of Schizophrenia.

Schizophrenia is a complex neuropsychiatric disorder with limited treatment options and highly debilitating symptoms, leading to poor personal, social, and occupational outcomes for an afflicted individual. Our current understanding of schizophrenia suggests that dopaminergic and glutamatergic systems have a significant role in the pathogenesis of the disease. Kynurenic acid, an endogenous glutamate antagonist, is found in elevated concentrations in the prefrontal cortex and cerebrospinal fluid of patients with schizophrenia, and this affects neurotransmitter release in a similar manner to previously observed psychotomimetic agents, such as phencyclidine, underlining the molecular basis to its link in schizophrenia pathophysiology. Kynurenic acid is a breakdown product of tryptophan degradation, through a transamination process mediated by kynurenine aminotransferase (KAT) enzymes. There are four KAT homologues reported, all of which are pyridoxal 5'- phosphate-dependent enzymes. All four KAT isoforms have been analysed structurally and biochemically, however the most extensive research is on KAT-I and KAT-II. These two enzymes have been targeted in structure-based drug design as a means of normalising raised kynurenic acid levels. The most potent KAT-I inhibitors and KAT-II inhibitors include phenylhydrazone hexanoic acid derivatives and a pyrazole series of compounds, respectively. KAT inhibitors have been shown to be effective in reducing kynurenic acid production, with accompanying changes in neurotransmitter release and pro-cognitive effects seen in animal studies. This review will discuss the characteristics pertaining to the different KAT isoforms, and will highlight the development of significant KAT inhibitors. KAT inhibitors have great potential for therapeutic application and represent a novel way in treating schizophrenia.

Homology modeling of human kynurenine aminotransferase III and observations on inhibitor binding using molecular docking.

Kynurenine aminotransferase (KAT) isozymes are responsible for catalyzing the conversion of kynurenine (KYN) to kynurenic acid (KYNA), which is considered to play a key role in central nervous system (CNS) disorders, including schizophrenia. The levels of KYNA in the postmortem prefrontal cortex and in the Cerebrospinal fluid (CSF) of schizophrenics are greater than normal brain. A basic strategy to decrease kynurenic acid levels is to promote the inhibition of the biosynthetic KAT isozymes. As there is no crystallographic model for human kynurenine aminotransferase III (KAT III), therefore, homology modeling has been performed based on the Mus musculus kynurenine aminotransferase III crystal structure (PDB ID: 3E2Y) as a template, and the model of the human KAT III was refined and optimized with molecular dynamics simulations. Further evaluation of the model quality was accomplished by investigating the interaction of KAT III inhibitors with the modeled enzyme. Such interactions were determined employing the AutoDock 4.2 program using the MGLTools 1.5.6 package. The most important interactions for the binding of the inhibitors, which are probably also central components of the active site of KAT III, were identified as Ala134, Tyr135, Lys 280, Lys 288, Thr285 and Arg429, which provide hydrogen bond interactions. Additionally, Tyr135 and Arg429 have good electrostatic interactions with inhibitors consistent with these residues also being essential for inhibition of the enzyme activity. We expect that this model and these docking data will be a useful resource for the rational design of novel drugs for treating neuropathologies.

A rare variant in human fibroblast activation protein associated with ER stress, loss of enzymatic function and loss of cell surface localisation.

Fibroblast activation protein (FAP) is a focus of interest as a potential cancer therapy target. This membrane bound protease possesses the unique catalytic activity of hydrolysis of the post-proline bond two or more residues from the N-terminus of substrates. FAP is highly expressed in activated fibroblastic cells in tumours, arthritis and fibrosis. A rare, novel, human polymorphism, C1088T, encoding Ser363 to Leu, occurring in the sixth blade of the ß propeller domain, was identified in a family. Both in primary human fibroblasts and in Ser363LeuFAP transfected cells, we showed that this single substitution ablates FAP dimerisation and causes loss of enzyme activity. Ser363LeuFAP was detectable only in endoplasmic reticulum (ER), in contrast to the distribution of wild-type FAP on the cell surface. The variant FAP showed decreased conformational antibody binding, consistent with an altered tertiary structure. Ser363LeuFAP expression was associated with upregulation of the ER chaperone BiP/GRP78, ER stress sensor ATF6, and the ER stress response target phospho-eIF2α, all indicators of ER stress. Proteasomal inhibition resulted in accumulation of Ser363LeuFAP, indicating the involvement of ER associated degradation (ERAD). Neither CHOP expression nor apoptosis was elevated, so ERAD is probably important for protecting Ser363LeuFAP expressing cells. These data on the first loss of function human FAP gene variant indicates that although the protein is vulnerable to an amino acid substitution in the ß-propeller domain, inactive, unfolded FAP can be tolerated by cells.

Design and synthesis of novel inhibitors of human kynurenine aminotransferase-I.

Herein we report 6-ethoxy-6-oxo-5-(2-phenylhydrazono) hexanoic acid and 3-(2-carboxyethyl)-1H-indole-2-carboxylic acid derivatives as synthetically accessible leads for human kynurenine aminotransferase-I (KAT-I) inhibitors. In total, 12 compounds were synthesized and their biological activities were determined using the HPLC-UV based KAT-I inhibition assay. Of the 12 compounds synthesized, 10 were found to inhibit human KAT-I and the most active compound was found to be 5-(2-(4-chlorophenyl) hydrazono)-6-ethoxy-6-oxohexanoic acid (9a) with an IC(50) of 19.8 µM.

Neuropeptide Y, B-type natriuretic peptide, substance P and peptide YY are novel substrates of fibroblast activation protein-α.

Fibroblast activation protein-α (FAP) is a cell surface-expressed and soluble enzyme of the prolyl oligopeptidase family, which includes dipeptidyl peptidase 4 (DPP4). FAP is not generally expressed in normal adult tissues, but is found at high levels in activated myofibroblasts and hepatic stellate cells in fibrosis and in stromal fibroblasts of epithelial tumours. FAP possesses a rare catalytic activity, hydrolysis of the post-proline bond two or more residues from the N-terminus of target substrates. α(2)-antiplasmin is an important physiological substrate of FAP endopeptidase activity. This study reports the first natural substrates of FAP dipeptidyl peptidase activity. Neuropeptide Y, B-type natriuretic peptide, substance P and peptide YY were the most efficiently hydrolysed substrates and the first hormone substrates of FAP to be identified. In addition, FAP slowly hydrolysed other hormone peptides, such as the incretins glucagon-like peptide-1 and glucose-dependent insulinotropic peptide, which are efficient DPP4 substrates. FAP showed negligible or no hydrolysis of eight chemokines that are readily hydrolysed by DPP4. This novel identification of FAP substrates furthers our understanding of this unique protease by indicating potential roles in cardiac function and neurobiology.

The dipeptidyl peptidase IV family in cancer and cell biology.

Of the 600+ known proteases identified to date in mammals, a significant percentage is involved or implicated in pathogenic and cancer processes. The dipeptidyl peptidase IV (DPIV) gene family, comprising four enzyme members [DPIV (EC 3.4.14.5), fibroblast activation protein, DP8 and DP9] and two nonenzyme members [DP6 (DPL1) and DP10 (DPL2)], are interesting in this regard because of their multiple diverse functions, varying patterns of distribution/localization and subtle, but significant, differences in structure/substrate recognition. In addition, their engagement in cell biological processes involves both enzymatic and nonenzymatic capabilities. This article examines, in detail, our current understanding of the biological involvement of this unique enzyme family and their overall potential as therapeutic targets.

Fibroblast activation protein and chronic liver disease.

Fibroblast activation protein (FAP) is the member of Dipeptidyl Peptidase IV (DPIV) gene family that is most similar to DPIV. Four members of this family, DPIV, FAP, DP8 and DP9 possess a rare catalytic activity, hydrolysis of a prolyl bond two residues from the substrate N terminus. Crystal structures show that the soluble form of FAP comprises two domains, an alpha/beta-hydrolase domain and an 8-blade beta-propeller domain. The interface between these two domains forms the catalytic pocket, and an opening for substrate access to the internal active site. The FAP homodimer is structurally very similar to DPIV but FAP glycoprotein expression is largely confined to mesenchymal cells in diseased and damaged tissue, notably the tissue remodelling region in chronically injured liver. FAP peptide substrates include denatured collagen and alpha2-antiplasmin. The functional roles of FAP in tumors and fibrotic tissue are not fully understood. This review places FAP in the context of chronic liver injury pathogenesis.