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1.
Nat Commun ; 10(1): 201, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30643150

RESUMO

Under hypoxia, most of glucose is converted to secretory lactate, which leads to the overuse of glutamine-carbon. However, under such a condition how glutamine nitrogen is disposed to avoid over-accumulating ammonia remains to be determined. Here we identify a metabolic flux of glutamine to secretory dihydroorotate, which is indispensable to glutamine-carbon metabolism under hypoxia. We found that glutamine nitrogen is necessary to nucleotide biosynthesis, but enriched in dihyroorotate and orotate rather than processing to its downstream uridine monophosphate under hypoxia. Dihyroorotate, not orotate, is then secreted out of cells. Furthermore, we found that the specific metabolic pathway occurs in vivo and is required for tumor growth. The identified metabolic pathway renders glutamine mainly to acetyl coenzyme A for lipogenesis, with the rest carbon and nitrogen being safely removed. Therefore, our results reveal how glutamine carbon and nitrogen are coordinatively metabolized under hypoxia, and provide a comprehensive understanding on glutamine metabolism.


Assuntos
Glutamina/metabolismo , Redes e Vias Metabólicas , Metaboloma , Neoplasias/metabolismo , Ácido Orótico/análogos & derivados , Acetilcoenzima A/metabolismo , Amônia/metabolismo , Amônia/toxicidade , Animais , Carbono/química , Carbono/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Glucose/metabolismo , Glutamina/química , Células HEK293 , Humanos , Ácido Láctico/metabolismo , Lipogênese , Metabolômica , Camundongos , Camundongos Nus , Neoplasias/sangue , Neoplasias/mortalidade , Neoplasias/patologia , Nitrogênio/química , Nitrogênio/metabolismo , Nucleotídeos/biossíntese , Ácido Orótico/metabolismo , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
2.
N Biotechnol ; 49: 104-111, 2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30347258

RESUMO

Nucleoside triphosphates (NTPs) are important synthetic targets with diverse applications in therapeutics and diagnostics. Enzymatic routes to NTPs from simple building blocks are attractive, however the cost and complexity of assembling the requisite mixtures of multiple enzymes hinders application. Here, we describe the use of an engineered E. coli cell-free lysate as an efficient readily-prepared multi-enzyme biocatalyst for the production of uridine triphosphate (UTP) from free ribose and nucleobase. Endogenous lysate enzymes are able to support the nucleobase ribosylation and nucleotide phosphorylation steps, while uridine phosphorylation and the production of ribose phosphates (ribose 1-phosphate, ribose 5-phosphate and phosphoribosyl pyrophosphate) require recombinant enrichment of endogenous activities. Co-expression vectors encoding all required recombinant enzymes were employed for host cell transformation, such that a cell-free lysate with all necessary activities was obtained from a single bacterial culture. ATP required as phosphorylation cofactor was recycled by endogenous lysate enzymes using cheap, readily-prepared acetyl phosphate. Surprisingly, acetyl phosphate initiated spontaneous generation of ATP in the lysate, most likely from the breakdown of endogenous pools of adenosine-containing starting materials (e.g. adenosine cofactors, ribonucleic acids). The sub-stoichiometric amount of ATP produced and recycled in this way was enough to support all ATP-dependent steps without addition of any exogenous cofactor or auxiliary enzyme. Using this approach, equimolar solutions of orotic acid and ribose are transformed near quantitatively into 1.4 g L-1 UTP within 2.5 h, using a low-cost, readily-generated biocatalytic preparation.


Assuntos
Trifosfato de Adenosina/farmacologia , Recombinação Genética , Ribose/metabolismo , Uracila/metabolismo , Uridina Trifosfato/biossíntese , Catálise , Escherichia coli/metabolismo , Hidrólise , Ácido Orótico/metabolismo , Recombinação Genética/genética , Ribose/química , Uracila/química , Uridina Trifosfato/química
3.
J Am Chem Soc ; 139(45): 16048-16051, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-29058891

RESUMO

Orotidine 5'-monophosphate decarboxylase (OMPDC) catalyzes the decarboxylation of 5-fluoroorotate (FO) with kcat/Km = 1.4 × 10-7 M-1 s-1. Combining this and related kinetic parameters shows that the 31 kcal/mol stabilization of the transition state for decarboxylation of OMP provided by OMPDC represents the sum of 11.8 and 10.6 kcal/mol stabilization by the substrate phosphodianion and the ribosyl ring, respectively, and an 8.6 kcal/mol stabilization from the orotate ring. The transition state for OMPDC-catalyzed decarboxylation of FO is stabilized by 5.2, 7.2, and 9.0 kcal/mol, respectively, by 1.0 M phosphite dianion, d-glycerol 3-phosphate and d-erythritol 4-phosphate. The stabilization is due to the utilization of binding interactions of the substrate fragments to drive an enzyme conformational change, which locks the orotate ring of the whole substrate, or the substrate pieces in a caged complex. We propose that enzyme-activation is a possible, and perhaps probable, consequence of any substrate-induced enzyme conformational change.


Assuntos
Ácido Orótico/análogos & derivados , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Biocatálise , Cinética , Modelos Moleculares , Conformação Molecular , Ácido Orótico/química , Ácido Orótico/metabolismo , Orotidina-5'-Fosfato Descarboxilase/química , Especificidade por Substrato
4.
Khirurgiia (Mosk) ; (3): 50-54, 2017.
Artigo em Russo | MEDLINE | ID: mdl-28374713

RESUMO

AIM: To study neocollagenogenesis after implantation of polypropylene endoprosthesis and polypropylene combined with polylactic acid endoprosthesis on background of «potassium orotate¼ administration. MATERIAL AND METHODS: We used two different types of endoprosthesis in the experiment. The first type was made of just polypropylene, the second type was made of polypropylene combined with polylactic acid. Histological examination was performed using polarizing microscopy. Collagen types I and III ratio in connective tissue around the prosthesis was analyzed according to the color that was individual for each type. RESULTS: The results were significantly better in case of collagenogenesis stimulation with Potassium orotate within 30 days and later for one type of endoprosthesis. Also we revealed that collagenogenesis and paraprosthesis capsule formation were more active in case of combined endoprosthesis. We revealed stimulating action of «Potassium Orotate¼ for collegenogenesis process, this fact was proved by increased collagen I/III ratio. CONCLUSION: Optimization of collagenogenesis was based on persistent 1,37-fold increase of collagen I/III ratio in case of combined endoprosthesis after 90 days. It was manifested by accelerated formation of connective tissue capsule and facilitated early isolation of the implant from surrounding tissues.


Assuntos
Colágeno/metabolismo , Tecido Conjuntivo , Implantes Experimentais , Ácido Orótico , Poliésteres/farmacologia , Polipropilenos/farmacologia , Implantação de Prótese/instrumentação , Regeneração/efeitos dos fármacos , Experimentação Animal , Animais , Materiais Biocompatíveis/metabolismo , Materiais Biocompatíveis/farmacologia , Disponibilidade Biológica , Tecido Conjuntivo/efeitos dos fármacos , Tecido Conjuntivo/metabolismo , Tecido Conjuntivo/patologia , Camundongos , Ácido Orótico/metabolismo , Ácido Orótico/farmacologia , Compostos de Potássio/metabolismo , Compostos de Potássio/farmacologia , Implantação de Prótese/métodos , Regeneração/fisiologia
5.
Biosci Biotechnol Biochem ; 81(6): 1227-1234, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28290772

RESUMO

We screened for factors involved in the cellulose-responsive induction of cellulose biomass-degrading enzyme genes from approximately 12,000 Aspergillus aculeatus T-DNA insertion mutants harboring a transcriptional fusion between the FIII-avicelase gene (cbhI) promoter and the orotidine 5'-monophosphate decarboxylase gene. Analysis of 5-fluoroorodic acid (5-FOA) sensitivity, cellulose utilization, and cbhI expression of the mutants revealed that a mutant harboring T-DNA at the dipeptidyl peptidase IV (dppIV) locus had acquired 5-FOA resistance and was deficient in cellulose utilization and cbhI expression. The deletion of dppIV resulted in a significant reduction in the cellulose-responsive expression of both cbhI as well as genes controlled by XlnR-independent and XlnR-dependent signaling pathways at an early phase in A. aculeatus. In contrast, the dppIV deletion did not affect the xylose-responsive expression of genes under the control of XlnR. These results demonstrate that DppIV participates in cellulose-responsive induction in A. aculeatus.


Assuntos
Aspergillus/genética , Celulases/genética , Celulose/metabolismo , Dipeptidil Peptidase 4/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Orotidina-5'-Fosfato Descarboxilase/genética , Aspergillus/efeitos dos fármacos , Aspergillus/enzimologia , Celulases/metabolismo , Celulose/farmacologia , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Dipeptidil Peptidase 4/agonistas , Dipeptidil Peptidase 4/metabolismo , Proteínas Fúngicas/metabolismo , Deleção de Genes , Mutagênese Insercional , Ácido Orótico/análogos & derivados , Ácido Orótico/metabolismo , Ácido Orótico/farmacologia , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Xilose/metabolismo , Xilose/farmacologia
6.
J Inherit Metab Dis ; 40(3): 423-431, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28205048

RESUMO

BACKGROUND: Elevated urinary excretion of orotic acid is associated with treatable disorders of the urea cycle and pyrimidine metabolism. Establishing the correct and timely diagnosis in a patient with orotic aciduria is key to effective treatment. Uridine monophosphate synthase is involved in de novo pyrimidine synthesis. Uridine monophosphate synthase deficiency (or hereditary orotic aciduria), due to biallelic mutations in UMPS, is a rare condition presenting with megaloblastic anemia in the first months of life. If not treated with the pyrimidine precursor uridine, neutropenia, failure to thrive, growth retardation, developmental delay, and intellectual disability may ensue. METHODS AND RESULTS: We identified mild and isolated orotic aciduria in 11 unrelated individuals with diverse clinical signs and symptoms, the most common denominator being intellectual disability/developmental delay. Of note, none had blood count abnormalities, relevant hyperammonemia or altered plasma amino acid profile. All individuals were found to have heterozygous alterations in UMPS. Four of these variants were predicted to be null alleles with complete loss of function. The remaining variants were missense changes and predicted to be damaging to the normal encoded protein. Interestingly, family screening revealed heterozygous UMPS variants in combination with mild orotic aciduria in 19 clinically asymptomatic family members. CONCLUSIONS: We therefore conclude that heterozygous UMPS-mutations can lead to mild and isolated orotic aciduria without clinical consequence. Partial UMPS-deficiency should be included in the differential diagnosis of mild orotic aciduria. The discovery of heterozygotes manifesting clinical symptoms such as hypotonia and developmental delay are likely due to ascertainment bias.


Assuntos
Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Orotato Fosforribosiltransferase/deficiência , Orotidina-5'-Fosfato Descarboxilase/deficiência , Erros Inatos do Metabolismo da Purina-Pirimidina/metabolismo , Anemia Megaloblástica/genética , Anemia Megaloblástica/metabolismo , Criança , Pré-Escolar , Feminino , Heterozigoto , Humanos , Lactente , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Masculino , Mutação/genética , Orotato Fosforribosiltransferase/genética , Orotato Fosforribosiltransferase/metabolismo , Ácido Orótico/metabolismo , Orotidina-5'-Fosfato Descarboxilase/genética , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Erros Inatos do Metabolismo da Purina-Pirimidina/genética , Pirimidinas/metabolismo , Distúrbios Congênitos do Ciclo da Ureia/genética , Distúrbios Congênitos do Ciclo da Ureia/metabolismo , Uridina/metabolismo
7.
Nucleosides Nucleotides Nucleic Acids ; 35(10-12): 566-577, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27906623

RESUMO

Orotate (OA) is well-known as a precursor in biosynthesis of pyrimidines; in mammals it is released from the mitochondrial dihydroorotate dehydrogenase (DHODH) for conversion to UMP by the cytoplasmic UMP synthase enzyme. OA is also a normal part of the diet, being found in milk and dairy products, and it is converted to uridine for use in the pyrimidine salvage pathway predominantly in liver, kidney and erythrocytes. Early research into nutrition identified orotate as "vitamin B13," and its use as a complex with organic cations or metal ions was promulgated in body-building, and in assisting therapies of metabolic syndromes. It has recently been established that the amelioration of gout by dairy products arises from the competition of orotate and urate at the hURAT1 transporter. The orotic aciduria that arises in children with defective UMP synthase can be rescued by oral uridine therapy, since UMP is the end-product and also a feedback inhibitor of the de novo pathway. In contrast, Miller (dysmorphology) syndrome is connected with defects in DHODH, and hence in the supply of OA, and cannot be helped by uridine. Other models of dysmorphisms are connected with enzymes early in the pyrimidine de novo pathway. We conclude that the OA molecule is itself required for the regulation of genes that are important in the development of cells, tissues and organisms.


Assuntos
Ácido Orótico/metabolismo , Animais , Anticolesterolemiantes/farmacologia , Anticolesterolemiantes/uso terapêutico , Dieta , Humanos , Ácido Orótico/farmacologia , Ácido Orótico/uso terapêutico
8.
Biochimie ; 131: 45-53, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27650727

RESUMO

De novo pyrimidine biosynthesis pathway is well developed and functional in protozoan parasite Leishmania donovani. The dihydroorotase (LdDHOase) is third enzyme of the pathway. The enzyme was cloned, expressed in E. coli BL21 (DE3), purified to homogeneity and biochemically characterized. The estimated kcat for the forward reaction and reverse reactions were 2.1 ± 0.1 s-1 and 1.1 ± 0.15 s-1, respectively. Homology modeling and docking studies were done to find out potential inhibitors for LdDHOase. Biotin sulfone and Kaempferol were found to be potential inhibitors of LdDHOase based on docking studies. These inhibitors were verified using recombinant LdDHOase and their anti-leishmanial effect was evaluated. Moreover, alterations in expressions of de novo as well as salvage pathways enzymes, after treatment of L. donovani with dihydroorotase inhibitor(s) were evaluated and discussed as survival mechanism of the pathogen. Further, effect of inhibition of cytidine deaminase, a key enzyme of salvage pathway of pyrimidine biosynthesis, was also evaluated on parasitic survival and alteration in gene expression of enzymes of both pathways. Further, effect of both pathways inhibition was also evaluated. The data suggests that the inhibition of single pathway can be overcome by increased expression of enzyme(s) of alternate pathway and both pathways seem to be equally important in the pathogen. When both pathways are simultaneously inhibited, parasite shows significant DNA damage and parasitic death.


Assuntos
Di-Hidro-Orotase/metabolismo , Leishmania donovani/metabolismo , Proteínas de Protozoários/metabolismo , Pirimidinas/metabolismo , Biotina/análogos & derivados , Biotina/química , Biotina/farmacologia , Citidina Desaminase/antagonistas & inibidores , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Di-Hidro-Orotase/antagonistas & inibidores , Di-Hidro-Orotase/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Quempferóis/química , Quempferóis/farmacologia , Cinética , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/genética , Simulação de Acoplamento Molecular , Estrutura Molecular , Ácido Orótico/análogos & derivados , Ácido Orótico/química , Ácido Orótico/metabolismo , Domínios Proteicos , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato , Sulfonas/química , Sulfonas/farmacologia
9.
Fungal Biol ; 120(9): 1146-55, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27567720

RESUMO

Of all of the natural polymers, lignin, an aromatic heteropolymer in plant secondary cell walls, is the most resistant to biological degradation. White-rot fungi are the only known organisms that can depolymerize or modify wood lignin. Investigating the mechanisms underlying lignin biodegradation by white-rot fungi would contribute to the ecofriendly utilization of woody biomass as renewable resources in the future. Efficient gene disruption, which is generally very challenging in the white-rot fungi, was established in Pleurotus ostreatus (the oyster mushroom). Some of the genes encoding manganese peroxidases, enzymes that are considered to be involved in lignin biodegradation, were disrupted separately, and the phenotype of each single-gene disruptant was analysed. However, it remains difficult to generate multi-gene disruptants in this fungus. Here we developed a new genetic transformation marker in P. ostreatus and demonstrated two marker recycling methods that use counter-selection to generate a multigene disruptant. This study will enable future genetic studies of white-rot fungi, and it will increase our understanding of the complicated mechanisms, which involve various enzymes, including lignin-degrading enzymes, underlying lignin biodegradation by these fungi.


Assuntos
Antifúngicos/metabolismo , Flucitosina/metabolismo , Ácido Orótico/análogos & derivados , Pleurotus/genética , Pleurotus/metabolismo , Seleção Genética , Proteínas Fúngicas/genética , Técnicas de Inativação de Genes/métodos , Marcadores Genéticos , Genética Microbiana/métodos , Ácido Orótico/metabolismo , Peroxidases/genética
10.
J Biosci Bioeng ; 122(6): 645-651, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27401770

RESUMO

Shewanella livingstonensis Ac10, a psychrotrophic bacterium isolated from Antarctic seawater, grows well at low temperatures close to 0°C. The bacterium is useful as a host in a low-temperature protein expression system. It is also useful as a model microorganism to investigate the mechanisms of microbial cold-adaptation. Versatile genetic manipulation techniques would be useful to investigate the biology of this bacterium and to develop its applications. In this study, we developed a method for targeted gene deletion and insertion by using the gene coding for orotidine-5'-phosphate decarboxylase (pyrF), which is involved in pyrimidine synthesis. We found that S. livingstonensis Ac10 is sensitive to 5-fluoroorotic acid (5-FOA), which is converted to a highly toxic compound by the product of pyrF. A uracil-auxotrophic strain resistant to 5-FOA was constructed by deleting pyrF, thus allowing the use of a plasmid-borne copy of pyrF for selection of recombinants. We constructed the pyrF complementation suicide plasmid pKKP, which contains pyrF, the R6K replication origin, the mob site of RP4, an antibiotic marker gene, and a multiple cloning site. To demonstrate pyrF-based gene replacement, we deleted the internal region of orf5, the gene coding for an eicosapentaenoic acid (EPA) synthesis enzyme. We also successfully inserted a His6-tag-coding sequence into orf8, the gene coding for another EPA synthesis enzyme. This system allows the markerless deletion and insertion of desired sequences at specific sites in the genome, which remarkably facilitates genetic manipulation of this bacterium.


Assuntos
Deleção de Genes , Marcação de Genes/métodos , Genes Bacterianos , Mutagênese Insercional/métodos , Mutagênese Sítio-Dirigida/métodos , Orotidina-5'-Fosfato Descarboxilase/genética , Shewanella/genética , Adaptação Biológica/genética , Sequência de Bases , Temperatura Baixa , Organismos Geneticamente Modificados , Ácido Orótico/análogos & derivados , Ácido Orótico/metabolismo , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Shewanella/enzimologia , Shewanella/metabolismo
11.
Gene ; 583(2): 102-111, 2016 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-26861612

RESUMO

Biosynthesis pathways of pyrimidine and purine are shown to play an important role in regular cellular activities. The biosynthesis can occur either through de novo or salvage pathways based on the requirement of the cell. The pyrimidine biosynthesis pathway has been linked to several disorders and various autoimmune diseases. Orotate phosphoribosyl transferase (OPRTase) is an important enzyme which catalyzes the conversion of orotate to orotate monophosphate in the fifth step of pyrimidine biosynthesis. Phylogenetic analysis of 228 OPRTase sequences shows the distribution of proteins across different living forms of life. High structural similarities between Thermusthermophilus and other organisms kindled us to concentrate on OPRTase as an anti-pathogenic target. In this study, a homology model of OPRTase was constructed using 2P1Z as a template. About 100 ns molecular dynamics simulation was performed to investigate the conformational stability and dynamic patterns of the protein. The amino acid residues (Met1, Asp2, Glu43, Ala44, Glu47, Lys51, Ala157 and Leu158) lining in the binding site were predicted using SiteMap. Further, structure based virtual screening was performed on the predicted binding site using ChemBridge, Asinex, Binding, NCI, TosLab and Zinc databases. Compounds retrieved from the screening collections were manually clustered. The resultant protein-ligand complexes were subjected to molecular dynamics simulations, which further validates the binding modes of the hits. The study may provide better insight for designing potent anti-pathogenic agent.


Assuntos
Proteínas de Bactérias/química , Inibidores Enzimáticos/química , Simulação de Dinâmica Molecular , Orotato Fosforribosiltransferase/química , Ácido Orótico/química , Thermus thermophilus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cinética , Ligantes , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Orotato Fosforribosiltransferase/antagonistas & inibidores , Orotato Fosforribosiltransferase/metabolismo , Ácido Orótico/metabolismo , Filogenia , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , Thermus thermophilus/química
12.
Sci Rep ; 6: 19141, 2016 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-26751736

RESUMO

The rising antibiotic resistance of bacteria imposes a severe threat on human health. Inhibition of bacterial virulence is an alternative approach to develop new antimicrobials. Molecules targeting antibiotic resistant enzymes have been used in combination with cognate antibiotics. It might be ideal that a molecule can simultaneously suppress virulence factors and antibiotic resistance. Here we combined genetic and computer-aided inhibitor screening to search for such molecules against the bacterial pathogen Pseudomonas aeruginosa. To identify target proteins that control both virulence and antibiotic resistance, we screened for mutants with defective cytotoxicity and biofilm formation from 93 transposon insertion mutants previously reported with increased antibiotic susceptibility. A pyrD mutant displayed defects in cytotoxicity, biofilm formation, quorum sensing and virulence in an acute mouse pneumonia model. Next, we employed a computer-aided screening to identify potential inhibitors of the PyrD protein, a dihydroorotate dehydrogenase (DHODase) involved in pyrimidine biosynthesis. One of the predicted inhibitors was able to suppress the enzymatic activity of PyrD as well as bacterial cytotoxicity, biofilm formation and antibiotic resistance. A single administration of the compound reduced the bacterial colonization in the acute mouse pneumonia model. Therefore, we have developed a strategy to identify novel treatment targets and antimicrobial molecules.


Assuntos
Antibacterianos/farmacologia , Descoberta de Drogas , Farmacorresistência Bacteriana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/patogenicidade , Animais , Antibacterianos/química , Biofilmes/efeitos dos fármacos , Modelos Animais de Doenças , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Camundongos , Testes de Sensibilidade Microbiana , Mutação , Ácido Orótico/metabolismo , Fenótipo , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/mortalidade , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Piocianina/biossíntese , Sistemas de Secreção Tipo III/genética , Uracila/metabolismo , Virulência/genética , Fatores de Virulência
13.
J Biosci Bioeng ; 121(6): 625-630, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26707627

RESUMO

We demonstrated that a Klura3Δ, mutant of the yeast Kluyveromyces lactis is able to produce and secrete into the growth medium considerable amounts of orotic acid. Using yeast extract-peptone-glucose (YPD) based media we optimized production conditions in flask and bioreactor cultures. With cells grown in YPD 5% glucose medium, the best production in flask was obtained with a 1:12.5 ratio for flask: culture volume, 180 rpm, 28°C and 200 mM MOPS for pH stabilization at neutral values (initial culture pH at 8.0). The best production in a 2 L bioreactor was achieved at 500 rpm with 1 vvm aeration, 28°C and pH 7.0. Under these optimum conditions, similar rates of orotic acid production were obtained and maximum concentration achieved after 96 h was 6.7 g/L in flask and bioreactor cultures. These results revealed an excellent reproducibility between both systems and provided evidence for the biotechnological potential of Klura3Δ strain to produce orotic acid since the amounts obtained are comparable to the production in flask using a similar mutant of the industrially valuable Corynebacterium glutamicum.


Assuntos
Reatores Biológicos/microbiologia , Kluyveromyces/genética , Kluyveromyces/metabolismo , Engenharia Metabólica , Mutação , Ácido Orótico/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biotecnologia , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Meios de Cultura/química , Meios de Cultura/metabolismo , Meios de Cultura/farmacologia , Concentração de Íons de Hidrogênio , Kluyveromyces/efeitos dos fármacos , Reprodutibilidade dos Testes , Temperatura Ambiente , Fatores de Tempo
14.
J Genet Genomics ; 42(5): 207-19, 2015 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-26059769

RESUMO

It is timely to consider the many facets of the small molecule orotic acid (OA), which is well-known as an essential intermediate of pyrimidine de novo synthesis. In addition, it can be taken up by erythrocytes and hepatocytes for conversion into uridine and for use in the pyrimidine recycling pathway. We discuss the link between dietary orotate and fatty liver in rats, and the potential for the alleviation of neonatal hyperbilirubinaemia. We address the development of orotate derivatives for application as anti-pyrimidine drugs, and of complexes with metal ions and organic cations to assist therapies of metabolic syndromes. Recent genetic data link human Miller syndrome to defects in the dihydroorotate dehydrogenase (DHODH) gene, hence to depleted orotate production. Another defect in pyrimidine biosynthesis, the orotic aciduria arising in humans and cattle with a deficiency of UMP synthase (UMPS), has different symptoms. More recent work leads us to conclude that OA may have a role in regulating gene transcription.


Assuntos
Ácido Orótico/metabolismo , Pirimidinas/biossíntese , Animais , Sistema Nervoso Central/metabolismo , Enzimas/metabolismo , Humanos , Leite/metabolismo
15.
Phys Chem Chem Phys ; 17(27): 17790-6, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26087682

RESUMO

The dihydroorotate dehydrogenase (DHOD) enzyme catalyzes the unique redox reaction in the de novo pyrimidine biosynthesis pathway. In this reaction, the oxidation of dihydroorotate (DHO) to orotate (OA) and reduction of the flavin mononucleotide (FMN) cofactor is catalysed by DHOD. The class 2 DHOD, to which the human enzyme belongs, was experimentally shown to follow a stepwise mechanism but the data did not allow the determination of the order of bond-breaking in a stepwise oxidation of DHO. The goal of this study is to understand the reaction mechanism at the molecular level of class 2 DHOD, which may aid in the design of inhibitors that selectively impact the activity of only certain members of the enzyme family. In this paper, the catalytic mechanism of oxidation of DHO to OA in human DHOD was studied using a hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) approach and Molecular Dynamics (MD) simulations. The free energy barriers calculated reveal that the mechanism in human DHOD occurs via a stepwise reaction pathway. In the first step, a proton is abstracted from the C5 of DHO to the deprotonated Ser215 side chain. Whereas, in the second step, the transfer of the hydride or hydride equivalent from the C6 of DHO to the N5 of FMN, where free energy barrier calculated by the DFT/MM level is 10.84 kcal mol(-1). Finally, a residual decomposition analysis was carried out in order to elucidate the influence of the catalytic region residues during DHO oxidation.


Assuntos
Simulação de Dinâmica Molecular , Ácido Orótico/análogos & derivados , Ácido Orótico/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Teoria Quântica , Sítios de Ligação , Biocatálise , Mononucleotídeo de Flavina/química , Humanos , Ácido Orótico/química , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Estrutura Terciária de Proteína , Termodinâmica
16.
Curr Microbiol ; 71(2): 229-34, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25917504

RESUMO

The impact of orotate accumulation in the medically important bacterium Pseudomonas aeruginosa was studied by deleting pyrE, the gene encoding orotate phosphoribosyltransferase and responsible for converting orotate into orotate monophosphate within the de novo pyrimidine synthesis pathway. The pyrE mutant accumulated orotate and exhibited decreased production of hemolysin, casein protease, and elastase. Feeding orotate at a concentration of 51.25 µM to the wild type, PAO1, likewise decreased production of these factors except for hemolysin, which was not affected. A significant increase in the pigments pyocyanin and pyoverdin was also observed. Pyocyanin increase in the pyrE mutant was heightened when the mutant was supplemented with orotate. Although pyoverdin production in the wild-type PAO1 was unaffected by orotate supplementation, a decrease in the mutant's production was observed when supplemented with orotate. These results indicate a significant reduction in virulence factor production in the pyrE mutant and reduction in some virulence factors in the wild type when supplemented with orotate.


Assuntos
Proteínas de Bactérias/metabolismo , Ácido Orótico/metabolismo , Pseudomonas aeruginosa/metabolismo , Pirimidinas/metabolismo , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Orotato Fosforribosiltransferase/genética , Orotato Fosforribosiltransferase/metabolismo , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Fatores de Virulência/genética
18.
Neuropediatrics ; 46(2): 123-5, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25757096

RESUMO

Hereditary orotic aciduria is a rare metabolic disease that results from a defect of uridine-5-monophosphate synthase (UMPS). In affected patients, main clinical symptoms are a markedly increased urinary excretion of orotic acid combined with megaloblastic anemia. This report describes a new case of UMPS deficiency without megaloblastic anemia but with epilepsy.


Assuntos
Anemia Megaloblástica/complicações , Epilepsia/complicações , Doenças Metabólicas/complicações , Complexos Multienzimáticos/deficiência , Orotato Fosforribosiltransferase/deficiência , Ácido Orótico/metabolismo , Orotidina-5'-Fosfato Descarboxilase/deficiência , Anemia Megaloblástica/metabolismo , Pré-Escolar , Epilepsia/metabolismo , Humanos , Masculino , Doenças Metabólicas/genética
19.
PLoS One ; 10(2): e0116594, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25647322

RESUMO

Ornithine transcarbamylase deficiency (OTCD, OMIM# 311250) is an inherited X-linked urea cycle disorder that is characterized by hyperammonemia and orotic aciduria. In this report, we describe a new animal model of OTCD caused by a spontaneous mutation in the mouse Otc gene (c.240T>A, p.K80N). This transversion in exon 3 of ornithine transcarbamylase leads to normal levels of mRNA with low levels of mature protein and is homologous to a mutation that has also been described in a single patient affected with late-onset OTCD. With higher residual enzyme activity, spf-J were found to have normal plasma ammonia and orotate. Baseline plasma amino acid profiles were consistent with mild OTCD: elevated glutamine, and lower citrulline and arginine. In contrast to WT, spf-J displayed baseline elevations in cerebral amino acids with depletion following immune challenge with polyinosinic:polycytidylic acid. Our results indicate that the mild spf-J mutation constitutes a new mouse model that is suitable for mechanistic studies of mild OTCD and the exploration of cerebral pathophysiology during acute decompensation that characterizes proximal urea cycle dysfunction in humans.


Assuntos
Aminoácidos/metabolismo , Encéfalo/metabolismo , Doença da Deficiência de Ornitina Carbomoiltransferase/imunologia , Doença da Deficiência de Ornitina Carbomoiltransferase/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Peso Corporal , Encéfalo/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Camundongos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Ornitina Carbamoiltransferase/química , Ornitina Carbamoiltransferase/genética , Ornitina Carbamoiltransferase/metabolismo , Doença da Deficiência de Ornitina Carbomoiltransferase/genética , Ácido Orótico/metabolismo , Fenótipo , Poli I-C/farmacologia , Estrutura Terciária de Proteína , Ratos , Análise de Sobrevida
20.
Phys Chem Chem Phys ; 15(43): 18863-71, 2013 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-24084894

RESUMO

Chagas' disease is considered to be a health problem affecting millions of people in Latin America. This disease is caused by the parasite Trypanosoma cruzi. Recently dihydroorotate dehydrogenase class 1A from Trypanosoma cruzi (TcDHODA) was shown to be essential for the survival and growth of T. cruzi and proposed as a drug target against Chagas' disease. This enzyme catalyzes the oxidation of (S)-dihydroorotate to orotate, with a proposed catalytic cycle consisting of two half-reactions. In the first half-reaction dihydroorotate is oxidized to orotate, with the consequent reduction of the flavin mononucleotide cofactor. In the second half-reaction fumarate is reduced to succinate. The first oxidation half-reaction may occur via a concerted or a stepwise mechanism. Herein, the catalytic mechanism of TcDHODA has been studied using hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) Molecular Dynamics (MD) simulations. The free energy profiles derived from the bidimensional potential of mean force reveal more details for two half-reaction processes.


Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Trypanosoma cruzi/enzimologia , Biocatálise , Ligações de Hidrogênio , Simulação de Dinâmica Molecular , Ácido Orótico/análogos & derivados , Ácido Orótico/química , Ácido Orótico/metabolismo , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Teoria Quântica , Eletricidade Estática
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