Monitoring type 2 diabetes from volatile faecal metabolome in Cushing's syndrome and single Afmid mouse models via a longitudinal study.

The analysis of volatile organic compounds (VOCs) as a non-invasive method for disease monitoring, such as type 2 diabetes (T2D) has shown potential over the years although not yet set in clinical practice. Longitudinal studies to date are limited and the understanding of the underlying VOC emission over the age is poorly understood. This study investigated longitudinal changes in VOCs present in faecal headspace in two mouse models of T2D - Cushing's syndrome and single Afmid knockout mice. Longitudinal changes in bodyweight, blood glucose levels and plasma insulin concentration were also reported. Faecal headspace analysis was carried out using selected ion flow tube mass spectrometry (SIFT-MS) and thermal desorption coupled to gas chromatography-mass spectrometry (TD-GC-MS). Multivariate data analysis of the VOC profile showed differences mainly in acetic acid and butyric acid able to discriminate the groups Afmid and Cushing's mice. Moreover, multivariate data analysis revealed statistically significant differences in VOCs between Cushing's mice/wild-type (WT) littermates, mainly short-chain fatty acids (SCFAs), ketones, and alcohols, and longitudinal differences mainly attributed to methanol, ethanol and acetone. Afmid mice did not present statistically significant differences in their volatile faecal metabolome when compared to their respective WT littermates. The findings suggested that mice developed a diabetic phenotype and that the altered VOC profile may imply a related change in gut microbiota, particularly in Cushing's mice. Furthermore, this study provided major evidence of age-related changes on the volatile profile of diabetic mice.

MYC promotes tryptophan uptake and metabolism by the kynurenine pathway in colon cancer.

Tumors display increased uptake and processing of nutrients to fulfill the demands of rapidly proliferating cancer cells. Seminal studies have shown that the proto-oncogene MYC promotes metabolic reprogramming by altering glutamine uptake and metabolism in cancer cells. How MYC regulates the metabolism of other amino acids in cancer is not fully understood. Using high-performance liquid chromatography (HPLC)-tandem mass spectrometry (LC-MS/MS), we found that MYC increased intracellular levels of tryptophan and tryptophan metabolites in the kynurenine pathway. MYC induced the expression of the tryptophan transporters SLC7A5 and SLC1A5 and the enzyme arylformamidase (AFMID), involved in the conversion of tryptophan into kynurenine. SLC7A5, SLC1A5, and AFMID were elevated in colon cancer cells and tissues, and kynurenine was significantly greater in tumor samples than in the respective adjacent normal tissue from patients with colon cancer. Compared with normal human colonic epithelial cells, colon cancer cells were more sensitive to the depletion of tryptophan. Blocking enzymes in the kynurenine pathway caused preferential death of established colon cancer cells and transformed colonic organoids. We found that only kynurenine and no other tryptophan metabolite promotes the nuclear translocation of the transcription factor aryl hydrocarbon receptor (AHR). Blocking the interaction between AHR and kynurenine with CH223191 reduced the proliferation of colon cancer cells. Therefore, we propose that limiting cellular kynurenine or its downstream targets could present a new strategy to reduce the proliferation of MYC-dependent cancer cells.

Cytotoxicity of trifluridine correlates with the thymidine kinase 1 expression level.

Trifluridine (FTD), a tri-fluorinated thymidine analogue, is a key component of the oral antitumor drug FTD/TPI (also known as TAS-102), which is used to treat refractory metastatic colorectal cancer. Thymidine kinase 1 (TK1) is thought to be important for the incorporation of FTD into DNA, resulting in DNA dysfunction and cytotoxicity. However, it remains unknown whether TK1 is essential for FTD incorporation into DNA and whether this event is affected by the expression level of TK1 because TK1-specific-deficient human cancer cell lines have not been established. Here, we generated TK1-knock-out human colorectal cancer cells using the CRISPR/Cas9 genome editing system and validated the specificity of TK1 knock-out by measuring expression of AFMID, which is encoded on the same locus as TK1. Using TK1-knock-out cells, we confirmed that TK1 is essential for cellular sensitivity to FTD. Furthermore, we demonstrated a correlation between the TK1 expression level and cytotoxicity of FTD using cells with inducible TK1 expression, which were generated from TK1-knock-out cells. Based on our finding that the TK1 expression level correlates with sensitivity to FTD, we suggest that FTD/TPI might efficiently treat cancers with high TK1 expression.

Genetic alterations affecting the genes encoding the enzymes of the kynurenine pathway and their association with human diseases.

Tryptophan is metabolized primarily via the kynurenine pathway (KP), which involves several enzymes, including indoleamine 2,3-dioxygenase, tryptophan 2,3 dioxygenase (TDO), kynurenine aminotransferases (KATs), kynurenine monooxygenase (KMO) etc. The majority of metabolites are neuroactive: some of them, such as kynurenic acid, show neuroprotective effects, while others contribute to free radical production, leading to neurodegeneration. Imbalance of the pathway is assumed to contribute to the development of several neurodegenerative diseases, psychiatric disorders, migraine and multiple sclerosis. Our aim was to summarize published data on genetic alterations of enzymes involved in the KP leading to disturbances of the pathway that can be related to different diseases. To achieve this, a PubMed literature search was performed for publications on genetic alterations of the KP enzymes upto April 2017. Several genetic alterations of the KP have been identified and have been proposed to be associated with diseases. Here we must emphasize that despite the large number of recognized genetic alterations, the number of firmly established causal relations with specific diseases is still small. The realization of this by those interested in the field is very important and finding such connections should be a major focus of related research. Polymorphisms of the genes encoding the enzymes of the KP have been associated with autism, multiple sclerosis and schizophrenia, and were shown to affect the immune response of patients with bacterial meningitis, just to mention a few. To our knowledge, this is the first comprehensive review of the genetic alterations of the KP enzymes. We believe that the identification of genetic alterations underlying diseases has great value regarding both treatment and diagnostics in precision medicine, as this work can promote the understanding of pathological mechanisms, and might facilitate medicinal chemistry approaches to substitute missing components or correct the disturbed metabolite balance of KP.

Chemoproteomic, biochemical and pharmacological approaches in the discovery of inhibitors targeting human α/ß-hydrolase domain containing 11 (ABHD11).

ABHD11 (α/ß-hydrolase domain containing 11) is a non-annotated enzyme belonging to the family of metabolic serine hydrolases (mSHs). Its natural substrates and products are unknown. Using competitive activity-based protein profiling (ABPP) to identify novel inhibitors of human (h)ABHD11, three compounds from our chemical library exhibited low nanomolar potency towards hABHD11. Competitive ABPP of various proteomes revealed fatty acid amide hydrolase (FAAH) as the sole off-target among the mSHs. Our fluorescent activity assays designed for natural lipase substrates revealed no activity of hABHD11 towards mono- or diacylglycerols. A broader profiling using para-nitrophenyl (pNP)-linked substrates indicated no amidase/protease, phosphatase, sulfatase, phospholipase C or phosphodiesterase activity. Instead, hABHD11 readily utilized para-nitrophenyl butyrate (pNPC4), indicating lipase/esterase-type activity that could be exploited in inhibitor discovery. Additionally, a homology model was created based on the crystal structure of bacterial esterase YbfF. In contrast to YbfF, which reportedly hydrolyze long-chain acyl-CoA, hABHD11 did not utilize oleoyl-CoA or arachidonoyl-CoA. In conclusion, the present study reports the discovery of potent hABHD11 inhibitors with good selectivity among mSHs. We developed substrate-based activity assays for hABHD11 that could be further exploited in inhibitor discovery and created the first homology-based hABHD11 model, offering initial insights into the active site of this poorly characterized enzyme.

Biochemical identification and crystal structure of kynurenine formamidase from Drosophila melanogaster.

KFase (kynurenine formamidase), also known as arylformamidase and formylkynurenine formamidase, efficiently catalyses the hydrolysis of NFK (N-formyl-L-kynurenine) to kynurenine. KFase is the second enzyme in the kynurenine pathway of tryptophan metabolism. A number of intermediates formed in the kynurenine pathway are biologically active and implicated in an assortment of medical conditions, including cancer, schizophrenia and neurodegenerative diseases. Consequently, enzymes involved in the kynurenine pathway have been considered potential regulatory targets. In the present study, we report, for the first time, the biochemical characterization and crystal structures of Drosophila melanogaster KFase conjugated with an inhibitor, PMSF. The protein architecture of KFase reveals that it belongs to the α/ß hydrolase fold family. The PMSF-binding information of the solved conjugated crystal structure was used to obtain a KFase and NFK complex using molecular docking. The complex is useful for understanding the catalytic mechanism of KFase. The present study provides a molecular basis for future efforts in maintaining or regulating kynurenine metabolism through the molecular and biochemical regulation of KFase.

Tryptophan catabolism via kynurenine production in Streptomyces coelicolor: identification of three genes coding for the enzymes of tryptophan to anthranilate pathway.

Most enzymes involved in tryptophan catabolism via kynurenine formation are highly conserved in Prokaryotes and Eukaryotes. In humans, alterations of this pathway have been related to different pathologies mainly involving the central nervous system. In Bacteria, tryptophan and some of its derivates are important antibiotic precursors. Tryptophan degradation via kynurenine formation involves two different pathways: the eukaryotic kynurenine pathway, also recently found in some bacteria, and the tryptophan-to-anthranilate pathway, which is widespread in microorganisms. The latter produces anthranilate using three enzymes also involved in the kynurenine pathway: tryptophan 2,3-dioxygenase (TDO), kynureninase (KYN), and kynurenine formamidase (KFA). In Streptomyces coelicolor, where it had not been demonstrated which genes code for these enzymes, tryptophan seems to be important for the calcium- dependent antibiotic (CDA) production. In this study, we describe three adjacent genes of S. coelicolor (SCO3644, SCO3645, and SCO3646), demonstrating their involvement in the tryptophan-to-anthranilate pathway: SCO3644 codes for a KFA, SCO3645 for a KYN and SCO3646 for a TDO. Therefore, these genes can be considered as homologous respectively to kynB, kynU, and kynA of other microorganisms and belong to a constitutive catabolic pathway in S. coelicolor, which expression increases during the stationary phase of a culture grown in the presence of tryptophan. Moreover, the S. coelicolor ΔkynU strain, in which SCO3645 gene is deleted, produces higher amounts of CDA compared to the wild-type strain. Overall, these results describe a pathway, which is used by S. coelicolor to catabolize tryptophan and that could be inactivated to increase antibiotic production.

The actinomycin biosynthetic gene cluster of Streptomyces chrysomallus: a genetic hall of mirrors for synthesis of a molecule with mirror symmetry.

A gene cluster was identified which contains genes involved in the biosynthesis of actinomycin encompassing 50 kb of contiguous DNA on the chromosome of Streptomyces chrysomallus. It contains 28 genes with biosynthetic functions and is bordered on both sides by IS elements. Unprecedentedly, the cluster consists of two large inverted repeats of 11 and 13 genes, respectively, with four nonribosomal peptide synthetase genes in the middle. Nine genes in each repeat have counterparts in the other, in the same arrangement but in the opposite orientation, suggesting an inverse duplication of one of the arms during the evolution of the gene cluster. All of the genes appear to be organized into operons, each corresponding to a functional section of actinomycin biosynthesis, such as peptide assembly, regulation, resistance, and biosynthesis of the precursor of the actinomycin chromophore 4-methyl-3-hydroxyanthranilic acid (4-MHA). For 4-MHA synthesis, functional analysis revealed genes that encode pathway-specific isoforms of tryptophan dioxygenase, kynurenine formamidase, and hydroxykynureninase, which are distinct from the corresponding enzyme activities of cellular tryptophan catabolism in their regulation and in part in their substrate specificity. Phylogenetic analysis indicates that the pathway-specific tryptophan metabolism in Streptomyces most probably evolved divergently from the normal pathway of tryptophan catabolism to provide an extra or independent supply of building blocks for the synthesis of tryptophan-derived secondary metabolites.

Cloning, expression, and catalytic triad of recombinant arylformamidase.

Arylformamidase (AFMID) is the second enzyme of the kynurenine pathway metabolizing tryptophan to nicotinic acid and nicotinamide adenine dinucleotide cofactors. Inhibition of AFMID by organophosphorus insecticides in developing chicken embryos is correlated with lowered NAD levels and severe teratogenesis. The cDNA sequence previously identified for mouse liver AFMID (AF399717) (MW 34229) was cloned and expressed in Escherichia coli. Residues identified as potential catalytic triad members (S162, D247, and H279) through sequence motif and homology modeling were mutated to alanine to probe their contributions to enzyme activity. The wild-type and mutant AFMIDs were expressed as amino terminal 6 x His-tagged recombinant proteins to facilitate purification. Three chromatography steps isolated highly purified proteins for enzyme activity comparisons. Expressed AFMID showed high activity, 42+/-1 micromol/min/mg protein, for its natural substrate, N-formyl-l-kynurenine. The same K(m) (0.18--0.19 mM) was observed for expressed and native cytosolic AFMID. The single mutants (S162A, D247A, and H279A) lost essentially all (>99%) activity. The predicted catalytic triad of S162, D247, and H279 is therefore confirmed by site-directed mutagenesis.

Effect of arylformamidase (kynurenine formamidase) gene inactivation in mice on enzymatic activity, kynurenine pathway metabolites and phenotype.

The gene coding for arylformamidase (Afmid, also known as kynurenine formamidase) was inactivated in mice through the removal of a shared bidirectional promoter region regulating expression of the Afmid and thymidine kinase (Tk) genes. Afmid/Tk -deficient mice are known to develop sclerosis of glomeruli and to have an abnormal immune system. Afmid-catalyzed hydrolysis of N-formyl-kynurenine is a key step in tryptophan metabolism and biosynthesis of kynurenine-derived products including kynurenic acid, quinolinic acid, nicotinamide, NAD, and NADP. A disruption of these pathways is implicated in neurotoxicity and immunotoxicity. In wild-type (WT) mice, Afmid-specific activity (as measured by formyl-kynurenine hydrolysis) was 2-fold higher in the liver than in the kidney. Formyl-kynurenine hydrolysis was reduced by approximately 50% in mice heterozygous (HZ) for Afmid/Tk and almost completely eliminated in Afmid/Tk knockout (KO) mice. However, there was 13% residual formyl-kynurenine hydrolysis in the kidney of KO mice, suggesting the existence of a formamidase other than Afmid. Liver and kidney levels of nicotinamide plus NAD/NADP remained the same in WT, HZ and KO mice. Plasma concentrations of formyl-kynurenine, kynurenine, and kynurenic acid were elevated in KO mice (but not HZ mice) relative to WT mice, further suggesting that there must be enzymes other than Afmid (possibly in the kidney) capable of metabolizing formyl-kynurenine into kynurenine. Gradual kidney deterioration and subsequent failure in KO mice is consistent with high levels of tissue-specific Afmid expression in the kidney of WT but not KO mice. On this basis, the most significant function of the kynurenine pathway and Afmid in mice may be in eliminating toxic metabolites and to a lesser extent in providing intermediates for other processes.

NAD biosynthesis: identification of the tryptophan to quinolinate pathway in bacteria.

Previous studies have demonstrated two different biosynthetic pathways to quinolinate, the universal de novo precursor to the pyridine ring of NAD. In prokaryotes, quinolinate is formed from aspartate and dihydroxyacetone phosphate; in eukaryotes, it is formed from tryptophan. It has been generally believed that the tryptophan to quinolinic acid biosynthetic pathway is unique to eukaryotes; however, this paper describes the use of comparative genome analysis to identify likely candidates for all five genes involved in the tryptophan to quinolinic acid pathway in several bacteria. Representative examples of each of these genes were overexpressed, and the predicted functions are confirmed in each case using unambiguous biochemical assays.

Alternate activation of two divergently transcribed mouse genes from a bidirectional promoter is linked to changes in histone modification.

Thymidine kinase (TK) is a growth factor-inducible enzyme that is highly expressed in proliferating mammalian cells. Expression of mouse TK mRNA is controlled by transcriptional and posttranscriptional mechanisms including antisense transcription. Here we report the identification of a novel gene that is divergently transcribed from the bidirectional TK promoter. This gene encodes kynurenine formamidase (KF), an enzyme of the tryptophan metabolism. Whereas the TK gene is induced upon interleukin-2-mediated activation of resting T cells, the KF gene becomes simultaneously repressed. The TK promoter is regulated by E2F, SP1, histone acetyltransferases, and deacetylases. The binding site for the growth-regulated transcription factor E2F is beneficial for TK promoter activity but not required for KF expression. In contrast, the SP1 binding site is crucial for transcription in both directions. Inhibition of histone deacetylases by trichostatin A leads to increased histone acetylation at the TK/KF promoter and thereby to selective activation of the TK promoter and simultaneous shut-off of KF expression. Similarly, TK gene activation by interleukin-2 is linked to histone hyperacetylation, whereas KF expression correlates with reduced histone acetylation. The KF gene is the rare example of a mammalian gene whose expression is linked to histone hypoacetylation at its promoter.

Kynurenine formamidase: determination of primary structure and modeling-based prediction of tertiary structure and catalytic triad.

Kynurenine formamidase (KFase) (EC 3.5.1.9) hydrolyzes N-formyl-L-kynurenine, an obligatory step in the conversion of tryptophan to nicotinic acid. Low KFase activity in chicken embryos, from inhibition by organophosphorus insecticides and their metabolites such as diazoxon, leads to marked developmental abnormalities. While KFase was purportedly isolated previously, the structure and residues important for catalysis and inhibition were not established. KFase was isolated here from mouse liver cytosol by (NH4)2SO4 precipitation and three FPLC steps (resulting in 221-fold increase in specific activity for N-formyl-L-kynurenine hydrolysis) followed by conversion to [3H]diethylphosphoryl-KFase and finally isolation by C4 reverse-phase high-performance liquid chromatography. Determination of tryptic fragment amino acid sequences and cDNA cloning produced a new 305-amino-acid protein sequence. Although an amidase by function, the primary structure of KFase lacks the amidase signature sequence and is more similar to esterases and lipases. Sequence profile analysis indicates KFase is related to the esterase/lipase/thioesterase family containing the conserved active-site serine sequence GXSXG. The alpha/beta-hydrolase fold is suggested for KFase by its primary sequence and predicted secondary conformation. A three-dimensional model based on the structures of homologous carboxylesterase EST2 and brefeldin A esterase implicates Ser162, Asp247 and His279 as the active site triad.

Genetics of formamidase-5 (brain formamidase) in the mouse: localization of the structural gene on chromosome 14.

A single formamidase, which is different from the formamidases found in other tissues occurs in the brains of mice. This enzyme is here called formamidase-5 and the gene symbol is designated For-5. Two alleles are recognized on the basis of their differential heat sensitivity:For-5b is relatively heat stable and is present in strain C57BL/6J, while For-5d is relatively heat sensitive and is present in strain DBA/2J. The heat sensitivity of formamidase-5 in 44 other inbred strains and substrains was tested and found to resemble that of C57BL/6J or DBA/2J. Thirty-six recombinant inbred strains derived from progenitors that differed at For-5 were studies to test for single-gene inheritance and linkage with other loci. Complete concordance was found with the esterase-10 locus (Es-10), indicating close linkage. The 99% upper confidence limit of the distance between For-5 and Es-10 is 3.7 centimorgans (cM). Es-10 is located on chromosome 14 about 19 cM from the centromere. An independent demonstration of linkage of For-5 with Es-10 and another chromosome 14 marker, hairless (hr), is provided by the finding that the HRS/J strain, which has been sibmated for 60 generations with forced heterozygosity at the hr locus, is cosegregating at For-5 and Es-10. A survey of 32 inbred strains and substrains revealed that the For-5d allele is associated with the Es-10b allele, and that the For-5b allele is associated with Es-10a and Es-10c. Formamidase-5 segregates as expected in the F2 generation of crosses between strains bearing For-5b and For-5d alleles. It is possible that this unique formamidase of the brain is involved in the metabolism of a neurotransmitter substance.

Biochemical and genetic characterization of kynurenine formamidase from Drosophila melanogaster.

The molecular weight forms of kynurenine formamidase were studied both gentically and biochemically. Formamidase I (native molecular weight 60,000) was purified using (NH4)2SO4 and pH fractionation, DEAE-cellulose chromatography at two different pH's, hydroxylapatite chromatography, and Sephadex G-100 gel filtration. Its subunit molecular weight, as determined by SDS gel electrophoresis, is 34,000, indicating that formamidase I is a dimer. Its Km is 1.87 X 10(-3) M. Its isoelectric point is pH 5.3. Its amino acid composition is reported. Formamidase II (native molecular weight 31,000) was partially purified using techniques similar to those above. Its Km is 2.31 X 10(-3) M. The response of formamidase activity to change in gene dosage was measured in segmental aneuploids generated in the second, third, and X chromosomes. Two separate chromosomal regions were identified which when present in extra dosage result in an elevation of the level of formamidase activity close to that predicted for the addition of a structural gene in a two-gene system. These tentative map positions were substantiated by demonstration that addition of one of the regions, 25A-27E, causes a 50% elevation in the relative amount of formamidase II. Addition of the other region, 91B-93F, causes a similar elevation in the relative amount of formamidase I. A model of the evolutionary origin of the two forms is presented, and the significance of these results to this model is discussed.