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1.
Sci Rep ; 9(1): 18779, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31827172

RESUMO

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.


Assuntos
Arilformamidase/metabolismo , Síndrome de Cushing/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Metaboloma , Compostos Orgânicos Voláteis/metabolismo , Animais , Arilformamidase/genética , Glicemia/metabolismo , Síndrome de Cushing/diagnóstico , Diabetes Mellitus Experimental/diagnóstico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/diagnóstico , Fezes , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Microbioma Gastrointestinal , Insulina/sangue , Estudos Longitudinais , Masculino , Camundongos , Camundongos Knockout , Monitorização Fisiológica , Análise Multivariada , Obesidade/metabolismo
2.
Genes Dev ; 33(17-18): 1236-1251, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31416966

RESUMO

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.


Assuntos
Neoplasias do Colo/fisiopatologia , Cinurenina/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Triptofano/metabolismo , Sistema ASC de Transporte de Aminoácidos/genética , Antineoplásicos/farmacologia , Arilformamidase/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Indóis/farmacologia , Cinurenina/genética , Transportador 1 de Aminoácidos Neutros Grandes/genética , Antígenos de Histocompatibilidade Menor/genética , Oximas/farmacologia , Sulfonamidas/farmacologia
3.
Sci Rep ; 9(1): 7964, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138881

RESUMO

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.


Assuntos
Arilformamidase/genética , Citotoxinas/farmacologia , Regulação Neoplásica da Expressão Gênica , Timidina Quinase/genética , Trifluridina/farmacologia , Arilformamidase/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxiciclina/farmacologia , Efeito Fundador , Deleção de Genes , Células HCT116 , Células HT29 , Humanos , Transdução de Sinais
4.
Angew Chem Int Ed Engl ; 58(25): 8394-8399, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-30963655

RESUMO

l-4-Chlorokynurenine (l-4-Cl-Kyn) is a neuropharmaceutical drug candidate that is in development for the treatment of major depressive disorder. Recently, this amino acid was naturally found as a residue in the lipopeptide antibiotic taromycin. Herein, we report the unprecedented conversion of l-tryptophan into l-4-Cl-Kyn catalyzed by four enzymes in the taromycin biosynthetic pathway from the marine bacterium Saccharomonospora sp. CNQ-490. We used genetic, biochemical, structural, and analytical techniques to establish l-4-Cl-Kyn biosynthesis, which is initiated by the flavin-dependent tryptophan chlorinase Tar14 and its flavin reductase partner Tar15. This work revealed the first tryptophan 2,3-dioxygenase (Tar13) and kynurenine formamidase (Tar16) enzymes that are selective for chlorinated substrates. The substrate scope of Tar13, Tar14, and Tar16 was examined and revealed intriguing promiscuity, thereby opening doors for the targeted engineering of these enzymes as useful biocatalysts.


Assuntos
Aminoácidos/metabolismo , Antibacterianos/metabolismo , Antidepressivos/metabolismo , Cinurenina/análogos & derivados , Lipopeptídeos/metabolismo , Pró-Fármacos/metabolismo , Aminoácidos/química , Antibacterianos/química , Antidepressivos/química , Arilformamidase/metabolismo , Cristalografia por Raios X , Cinurenina/biossíntese , Cinurenina/química , Lipopeptídeos/química , Modelos Moleculares , Estrutura Molecular , Pró-Fármacos/química , Triptofano Oxigenase/metabolismo
5.
Sci Rep ; 8(1): 13104, 2018 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-30166577

RESUMO

The catalytic mechanism of the cyclic amidohydrolase isatin hydrolase depends on a catalytically active manganese in the substrate-binding pocket. The Mn2+ ion is bound by a motif also present in other metal dependent hydrolases like the bacterial kynurenine formamidase. The crystal structures of the isatin hydrolases from Labrenzia aggregata and Ralstonia solanacearum combined with activity assays allow for the identification of key determinants specific for the reaction mechanism. Active site residues central to the hydrolytic mechanism include a novel catalytic triad Asp-His-His supported by structural comparison and hybrid quantum mechanics/classical mechanics simulations. A hydrolytic mechanism for a Mn2+ dependent amidohydrolases that disfavour Zn2+ as the primary catalytically active site metal proposed here is supported by these likely cases of convergent evolution. The work illustrates a fundamental difference in the substrate-binding mode between Mn2+ dependent isatin hydrolase like enzymes in comparison with the vast number of Zn2+ dependent enzymes.


Assuntos
Amidoidrolases/metabolismo , Proteínas de Bactérias/metabolismo , Biocatálise , Manganês/metabolismo , Rhodobacteraceae/enzimologia , Zinco/metabolismo , Amidoidrolases/química , Sequência de Aminoácidos , Arilformamidase/metabolismo , Proteínas de Bactérias/química , Domínio Catalítico , Sequência Conservada , Evolução Molecular , Glutamina/metabolismo , Hidrólise , Isatina/química , Isatina/metabolismo , Cinurenina/metabolismo , Modelos Moleculares , Prótons , Teoria Quântica
6.
Mutat Res Rev Mutat Res ; 776: 32-45, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29807576

RESUMO

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.


Assuntos
Cinurenina/metabolismo , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , 3-Hidroxiantranilato 3,4-Dioxigenase/genética , 3-Hidroxiantranilato 3,4-Dioxigenase/metabolismo , Arilformamidase/genética , Arilformamidase/metabolismo , Carboxiliases/genética , Carboxiliases/metabolismo , Humanos , Hidrolases/genética , Hidrolases/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Quinurenina 3-Mono-Oxigenase/genética , Quinurenina 3-Mono-Oxigenase/metabolismo , Redes e Vias Metabólicas/genética , Transaminases/genética , Transaminases/metabolismo , Triptofano/metabolismo , Triptofano Oxigenase/genética , Triptofano Oxigenase/metabolismo
7.
Toxicol In Vitro ; 44: 357-360, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28782636

RESUMO

The objective of this study was to provide in ovo evidence for the proposed role of kynurenine formamidase of l-tryptophan to NAD+ pathway in embryo yolk sac membranes as a primary target for organophosphorus insecticide (OPI) teratogens in OPI-induced NAD-associated avian teratogenesis. Slices prepared from yolk sac membranes or embryo livers of chicken eggs treated with the OPI dicrotophos and/or methyl parathion were incubated with l-tryptophan. Yolk sac membrane slices metabolized l-tryptophan in the pathway to NAD+ before that function was established in livers. OPI interfered in ovo with the second step of l-tryptophan to NAD+ biosynthesis by inhibiting kynurenine formamidase. Its inhibition due to the teratogen dicrotophos occurred in yolk sac membranes during the period of embryo highest susceptibility to OPI teratogens in contrast to delayed and lower inhibition caused by the nonteratogen methyl parathion. Both OPI affected liver kynurenine formamidase in a similar manner. The onsets of liver enzyme inhibition, however, were delayed by about two days and occurred at the time of the reduced embryo susceptibility to teratogens. The early disruption of l-tryptophan metabolism and higher inhibition of kynurenine formamidase in yolk sac membranes may be the factors that determine action of OPI as teratogens in chicken embryos.


Assuntos
Arilformamidase/metabolismo , Inseticidas/toxicidade , Metil Paration/toxicidade , Compostos Organofosforados/toxicidade , Teratógenos/toxicidade , Triptofano/metabolismo , Animais , Embrião de Galinha , Cinurenina/análogos & derivados , Cinurenina/metabolismo , Fígado/metabolismo , NAD/metabolismo , Teratogênese , Saco Vitelino
8.
Eur J Pharm Sci ; 93: 253-63, 2016 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-27544863

RESUMO

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.


Assuntos
Serina Proteases/metabolismo , Inibidores de Serino Proteinase/farmacologia , Animais , Arilformamidase/genética , Encéfalo/metabolismo , Linhagem Celular Tumoral , Descoberta de Drogas , Feminino , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Modelos Moleculares , Proteômica , Serina Proteases/química , Serina Proteases/genética , Tioléster Hidrolases/genética
9.
Res Microbiol ; 167(3): 159-67, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26654915

RESUMO

Kynurenine formamidase (KynB) forms part of the kynurenine pathway which metabolises tryptophan to anthranilate. This metabolite can be used for downstream production of 2-alkyl-4-quinolone (AQ) signalling molecules that control virulence in Pseudomonas aeruginosa. Here we investigate the role of kynB in the production of AQs and virulence-associated phenotypes of Burkholderia pseudomallei K96243, the causative agent of melioidosis. Deletion of kynB resulted in reduced AQ production, increased biofilm formation, decreased swarming and increased tolerance to ciprofloxacin. Addition of exogenous anthranilic acid restored the biofilm phenotype, but not the persister phenotype. This study suggests the kynurenine pathway is a critical source of anthranilate and signalling molecules that may regulate B. pseudomallei virulence.


Assuntos
Arilformamidase/metabolismo , Biofilmes/crescimento & desenvolvimento , Burkholderia pseudomallei/enzimologia , Burkholderia pseudomallei/fisiologia , Locomoção , Quinolonas/metabolismo , Transdução de Sinais , Burkholderia pseudomallei/genética , Deleção de Genes , Triptofano/metabolismo , Virulência , ortoaminobenzoatos/metabolismo
10.
Biochem J ; 462(3): 581-9, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-24942958

RESUMO

Tryptophan is an important precursor for chemical entities that ultimately support the biosynthesis of key metabolites. The second stage of tryptophan catabolism is catalysed by kynurenine formamidase, an enzyme that is different between eukaryotes and prokaryotes. In the present study, we characterize the catalytic properties and present the crystal structures of three bacterial kynurenine formamidases. The structures reveal a new amidase protein fold, a highly organized and distinctive binuclear Zn2+ catalytic centre in a confined, hydrophobic and relatively rigid active site. The structure of a complex with 2-aminoacetophenone delineates aspects of molecular recognition extending to the observation that the substrate itself may be conformationally restricted to assist binding in the confined space of the active site and for subsequent processing. The cations occupy a crowded environment, and, unlike most Zn2+-dependent enzymes, there is little scope to increase co-ordination number during catalysis. We propose that the presence of a bridging water/hydroxide ligand in conjunction with the placement of an active site histidine supports a distinctive amidation mechanism.


Assuntos
Arilformamidase/química , Zinco/metabolismo , Arilformamidase/metabolismo , Bacillus anthracis/enzimologia , Biocatálise , Burkholderia cenocepacia/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Cinética , Conformação Proteica , Pseudomonas aeruginosa/enzimologia
11.
Environ Toxicol Pharmacol ; 35(2): 200-6, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23328119

RESUMO

This paper provides new information related to the mechanism of OPI (organophosphorus insecticides) teratogenesis. The COMFA (comparative molecular field analysis) and COMSIA (comparative molecular similarity indices analysis) suggest that the electrostatic and steric fields are the best predictors of OPI structural requirements to inhibit in ovo chicken embryo yolk sac membrane kynurenine formamidase, the proposed target for OPI teratogens. The dominant electrostatic interactions are localized at nitrogen-1, nitrogen-3, nitrogen of 2-amino substituent of the pyrimidinyl of pyrimidinyl phosphorothioates, and the oxygen of crotonamide carbonyl in crotonamide phosphates. Bulkiness of the substituents at carbon-2 and carbon-6 of the pyrimidinyls and/or N-substituents and carbon-3 substituents of crotonamides are the steric structural components that contribute to superiority of those OPI as in ovo inhibitors of kynurenine formamidase.


Assuntos
Arilformamidase/antagonistas & inibidores , Inseticidas/química , Inseticidas/toxicidade , Compostos Organofosforados/química , Compostos Organofosforados/toxicidade , Relação Quantitativa Estrutura-Atividade , Teratógenos/toxicidade , Saco Vitelino/enzimologia , Animais , Embrião de Galinha , Inibidores Enzimáticos/química , Inibidores Enzimáticos/toxicidade , Leptofós/química , Leptofós/toxicidade , Modelos Moleculares , Paration/química , Paration/toxicidade , Eletricidade Estática , Teratógenos/química , Saco Vitelino/efeitos dos fármacos
12.
Biochem J ; 446(2): 253-60, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22690733

RESUMO

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.


Assuntos
Arilformamidase/química , Arilformamidase/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Animais , Arilformamidase/antagonistas & inibidores , Arilformamidase/genética , Biocatálise , Domínio Catalítico , Simulação por Computador , Cristalografia por Raios X , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Inseticidas/química , Inseticidas/metabolismo , Inseticidas/farmacologia , Cinética , Cinurenina/análogos & derivados , Cinurenina/química , Cinurenina/metabolismo , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Compostos de Tosil/química , Compostos de Tosil/metabolismo , Compostos de Tosil/farmacologia
13.
Appl Microbiol Biotechnol ; 94(3): 719-28, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22234533

RESUMO

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.


Assuntos
Arilformamidase/genética , Hidrolases/genética , Cinurenina/metabolismo , Redes e Vias Metabólicas/genética , Streptomyces coelicolor/genética , Triptofano Oxigenase/genética , Triptofano/metabolismo , Arilformamidase/metabolismo , Hidrolases/metabolismo , Streptomyces coelicolor/metabolismo , Triptofano Oxigenase/metabolismo
14.
J Bacteriol ; 192(10): 2583-95, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20304989

RESUMO

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.


Assuntos
Proteínas de Bactérias/genética , Dactinomicina/biossíntese , Dactinomicina/química , Família Multigênica/genética , Isoformas de Proteínas/genética , Streptomyces/metabolismo , Arilformamidase/genética , Arilformamidase/metabolismo , Proteínas de Bactérias/metabolismo , Cromatografia em Camada Delgada , Dactinomicina/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Isoformas de Proteínas/metabolismo , Análise de Sequência de DNA , Estereoisomerismo , Streptomyces/genética , ortoaminobenzoatos/metabolismo
15.
Biochemistry ; 47(6): 1608-21, 2008 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-18205391

RESUMO

The essential enzymatic cofactor NAD+ can be synthesized in many eukaryotes, including Saccharomyces cerevisiae and mammals, using tryptophan as a starting material. Metabolites along the pathway or on branches have important biological functions. For example, kynurenic acid can act as an NMDA antagonist, thereby functioning as a neuroprotectant in a wide range of pathological states. N-Formyl kynurenine formamidase (FKF) catalyzes the second step of the NAD+ biosynthetic pathway by hydrolyzing N-formyl kynurenine to produce kynurenine and formate. The S. cerevisiae FKF had been reported to be a pyridoxal phosphate-dependent enzyme encoded by BNA3. We used combined crystallographic, bioinformatic and biochemical methods to demonstrate that Bna3p is not an FKF but rather is most likely the yeast kynurenine aminotransferase, which converts kynurenine to kynurenic acid. Additionally, we identify YDR428C, a yeast ORF coding for an alpha/beta hydrolase with no previously assigned function, as the FKF. We predicted its function based on our interpretation of prior structural genomics results and on its sequence homology to known FKFs. Biochemical, bioinformatics, genetic and in vivo metabolite data derived from LC-MS demonstrate that YDR428C, which we have designated BNA7, is the yeast FKF.


Assuntos
Arilformamidase/metabolismo , Saccharomyces cerevisiae/enzimologia , Transaminases/metabolismo , Sequência de Aminoácidos , Arilformamidase/química , Cromatografia Líquida de Alta Pressão , Biologia Computacional , Cristalografia , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Transaminases/química
16.
Protein Expr Purif ; 44(1): 39-44, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15935693

RESUMO

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.


Assuntos
Substituição de Aminoácidos/genética , Arilformamidase/genética , Clonagem Molecular , Expressão Gênica , Fígado/enzimologia , Animais , Arilformamidase/isolamento & purificação , Arilformamidase/metabolismo , Sítios de Ligação/genética , Clonagem Molecular/métodos , Escherichia coli/genética , Expressão Gênica/genética , Camundongos , Mutagênese Sítio-Dirigida/métodos , Mutação Puntual/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
17.
Biochim Biophys Acta ; 1724(1-2): 163-72, 2005 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-15866519

RESUMO

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.


Assuntos
Arilformamidase/genética , Arilformamidase/metabolismo , Cinurenina/metabolismo , Animais , Arilformamidase/análise , Inativação Gênica , Rim/química , Rim/enzimologia , Cinurenina/sangue , Fígado/química , Fígado/enzimologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Niacinamida/análise , Fenótipo , Insuficiência Renal/genética , Timidina Quinase/análise , Timidina Quinase/genética , Triptofano/sangue , Triptofano/metabolismo
18.
FEMS Microbiol Lett ; 227(2): 219-27, 2003 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-14592712

RESUMO

While a variety of chemical transformations related to the aerobic degradation of L-tryptophan (kynurenine pathway), and most of the genes and corresponding enzymes involved therein have been predominantly characterized in eukaryotes, relatively little was known about this pathway in bacteria. Using genome comparative analysis techniques we have predicted the existence of the three-step pathway of aerobic L-tryptophan degradation to anthranilate (anthranilate pathway) in several bacteria. Based on the chromosomal gene clustering analysis, we have identified a previously unknown gene encoding for kynurenine formamidase (EC 3.5.1.19) involved with the second step of the anthranilate pathway. This functional prediction was experimentally verified by cloning, expression and enzymatic characterization of recombinant kynurenine formamidase orthologs from Bacillus cereus, Pseudomonas aeruginosa and Ralstonia metallidurans. Experimental verification of the inferred anthranilate pathway was achieved by functional expression in Escherichia coli of the R. metallidurans putative kynBAU operon encoding three required enzymes: tryptophan 2,3-dioxygenase (gene kynA), kynurenine formamidase (gene kynB), and kynureninase (gene kynU). Our data provide the first experimental evidence of the connection between these genes (only one of which, kynU, was previously characterized) and L-tryptophan aerobic degradation pathway in bacteria.


Assuntos
Arilformamidase/metabolismo , Bactérias/enzimologia , Triptofano/metabolismo , Aerobiose , Sequência de Aminoácidos , Bactérias/metabolismo , Genoma Bacteriano , Dados de Sequência Molecular , Óperon , ortoaminobenzoatos/metabolismo
19.
J Biol Chem ; 278(3): 1784-93, 2003 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-12411446

RESUMO

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.


Assuntos
Arilformamidase/genética , Regulação Enzimológica da Expressão Gênica , Histonas/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Timidina Quinase/genética , Acetilação , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Camundongos , Dados de Sequência Molecular , Transcrição Genética , Ativação Transcricional
20.
Chem Biol ; 10(12): 1195-204, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14700627

RESUMO

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.


Assuntos
Bactérias/enzimologia , Bactérias/genética , NAD/biossíntese , Ácido Quinolínico/metabolismo , Triptofano/metabolismo , Arilformamidase/genética , Arilformamidase/metabolismo , Bactérias/metabolismo , Cromossomos Bacterianos/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Teste de Complementação Genética , Hidrolases/genética , Hidrolases/metabolismo , Cinética , Dados de Sequência Molecular , NAD/química , Oxirredutases/genética , Oxirredutases/metabolismo , Ácido Quinolínico/química , Triptofano/química
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