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1.
Bioresour Technol ; 310: 123458, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32380436

RESUMO

Cell growth of Trichoderma reesei is greatly inhibited by furan derivatives (furfural and HMF) generated during pretreatment of lignocellulose, and the cellulase production is hence suppressed. In this study, a novel recombinant strain of T. reesei with high tolerance to furans was constructed by homologously co-expressing nicotinate phosphoribosyltransferase and alcohol dehydrogenase. We observed that furfural had a stronger inhibitory effect than HMF and cellulase production was decreased by 35% in T. reesei with the stress of 2.5 mM furfural. The activities of nicotinate phosphoribosyltransferase and alcohol dehydrogenase increased 8.6-fold and 2.9-fold in the recombinant strain, respectively. Furfural was effectively converted into furfuryl alcohol which was then depleted, thus the production of cellulase could be recovered when the recombinant strain was grown in 5% (w/v) two-step stem explosion pretreated rice straw without detoxification. This work presents an important strategy for efficient enzyme production in T. reesei from non-detoxified pretreated lignocellulose feedstocks.


Assuntos
Celulase , Trichoderma , Álcool Desidrogenase , Lignina , Pentosiltransferases
2.
Science ; 368(6496): 1211-1219, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32327601

RESUMO

The arabinosyltransferases EmbA, EmbB, and EmbC are involved in Mycobacterium tuberculosis cell wall synthesis and are recognized as targets for the anti-tuberculosis drug ethambutol. In this study, we determined cryo-electron microscopy and x-ray crystal structures of mycobacterial EmbA-EmbB and EmbC-EmbC complexes in the presence of their glycosyl donor and acceptor substrates and with ethambutol. These structures show how the donor and acceptor substrates bind in the active site and how ethambutol inhibits arabinosyltransferases by binding to the same site as both substrates in EmbB and EmbC. Most drug-resistant mutations are located near the ethambutol binding site. Collectively, our work provides a structural basis for understanding the biochemical function and inhibition of arabinosyltransferases and the development of new anti-tuberculosis agents.


Assuntos
Antituberculosos/química , Parede Celular/enzimologia , Etambutol/química , Mycobacterium tuberculosis/enzimologia , Pentosiltransferases/química , Microscopia Crioeletrônica , Farmacorresistência Bacteriana Múltipla , Conformação Proteica
3.
Bioresour Technol ; 307: 123258, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32247276

RESUMO

In this work, a mono- and a bi-enzymatic analytical immobilized enzyme reactors (IMERs) were developed as prototypes for biosynthetic purposes and their performances in the in-flow synthesis of nucleoside analogues of pharmaceutical interest were evaluated. Two biocatalytic routes based on nucleoside 2'-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) and uridine phosphorylase from Clostridium perfrigens (CpUP)/purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP) were investigated in the synthesis of 2'-deoxy, 2',3'-dideoxy and arabinonucleoside derivatives. LrNDT-IMER catalyzed the synthesis of 5-fluoro-2'-deoxyuridine and 5-iodo-2'-deoxyuridine in 65-59% conversion yield, while CpUP/AhPNP-IMER provided the best results for the preparation of arabinosyladenine (60% conversion yield). Both IMERs proved to be promising alternatives to chemical routes for the synthesis of nucleoside analogues. The developed in-flow system represents a powerful tool for the fast production on analytical scale of nucleosides for preliminary biological tests.


Assuntos
Enzimas Imobilizadas , Nucleosídeos , Biocatálise , Pentosiltransferases , Purina-Núcleosídeo Fosforilase
4.
Mutat Res ; 850-851: 503136, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32247553

RESUMO

Tumorigenesis induced by oxidative stress is thought to be initiated by mutagenesis, but via an indirect mechanism. The dose-response curves for agents that act by this route usually show a threshold, for unknown reasons. To gain insight into these phenomena, we have analyzed the dose response for mutagenesis induced by the oral administration of potassium bromate, a typical oxidative-stress-generating agent, to gpt delta mice. The agent was given orally for 90 d to either Nrf2+ or Nrf2-knockout (KO) mice and mutants induced in the small intestine were analyzed. In Nrf2+mice, the mutant frequency was significantly greater than in the vehicle controls at a dose of 0.6 g/L but not at 0.2 g/L, indicating that a practical threshold for mutagenesis lies between these doses. At 0.6 g/L, the frequencies of G-to-T transversions (landmark mutations for oxidative stress) and G-to-A transitions were significantly elevated. In Nrf2-KO mice, too, the total mutant frequency was increased only at 0.6 g/L. G-to-T transversions are likely to have driven tumorigenesis in the small intestine. A site-specific G-to-T transversion at guanine (nucleotide 406) in a 5'-TGAA-3' sequence in gpt, and our primer extension reaction showed that formation of the oxidative DNA base modification 8-oxo-deoxyguanosine (8-oxo-dG) at nucleotide 406 was significantly increased at doses of 0.6 and 2 g/L in the gpt delta mice. In the Apc oncogene, guanine residues in the same or similar sequences (TGAA or AGAA) are highly substituted by thymine (G-to-T transversions) in potassium bromate-induced tumors. We propose that formation of 8-oxo-dG in the T(A)GAA sequence is an initiating event in tumor formation in the small intestine in response to oxidative stress.


Assuntos
Bromatos/toxicidade , Mutagênese/genética , Estresse Oxidativo/genética , Pentosiltransferases/genética , 8-Hidroxi-2'-Desoxiguanosina/genética , Administração Oral , Animais , Bromatos/farmacologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , DNA/efeitos dos fármacos , DNA/genética , Relação Dose-Resposta a Droga , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/patologia , Camundongos , Camundongos Knockout , Mutagênese/efeitos dos fármacos , Mutação , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos
5.
Cell Host Microbe ; 27(1): 79-92.e9, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31901520

RESUMO

Efficient nutrient acquisition in the human gut is essential for microbial persistence. Although polysaccharides have been well-studied nutrients for the gut microbiome, other resources such as nucleic acids and nucleosides are less studied. We describe several ribose-utilization systems (RUSs) that are broadly represented in Bacteroidetes and appear to have diversified to access ribose from a variety of substrates. One Bacteroides thetaiotaomicron RUS variant is critical for competitive gut colonization in a diet-specific fashion. We used molecular genetics to probe the required functions of the system and the nature of the nutrient source(s) underlying this phenotype. Two RUS-encoded ribokinases were the only components required for this effect, presumably because they generate ribose-phosphate derivatives from products of an unlinked but essential nucleoside phosphorylase. Our results underscore the extensive mechanisms that gut symbionts have evolved to access nutrients and the potential for unexpected dependencies among systems that mediate colonization and persistence.


Assuntos
Bacteroides thetaiotaomicron , Pentosiltransferases/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Ribose/metabolismo , Animais , Bacteroides thetaiotaomicron/genética , Bacteroides thetaiotaomicron/metabolismo , Dieta , Microbioma Gastrointestinal/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Camundongos , Pentosiltransferases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Simbiose
6.
Nat Commun ; 11(1): 303, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949166

RESUMO

α-Dystroglycan (α-DG) is a highly-glycosylated surface membrane protein. Defects in the O-mannosyl glycan of α-DG cause dystroglycanopathy, a group of congenital muscular dystrophies. The core M3 O-mannosyl glycan contains tandem ribitol-phosphate (RboP), a characteristic feature first found in mammals. Fukutin and fukutin-related protein (FKRP), whose mutated genes underlie dystroglycanopathy, sequentially transfer RboP from cytidine diphosphate-ribitol (CDP-Rbo) to form a tandem RboP unit in the core M3 glycan. Here, we report a series of crystal structures of FKRP with and without donor (CDP-Rbo) and/or acceptor [RboP-(phospho-)core M3 peptide] substrates. FKRP has N-terminal stem and C-terminal catalytic domains, and forms a tetramer both in crystal and in solution. In the acceptor complex, the phosphate group of RboP is recognized by the catalytic domain of one subunit, and a phosphate group on O-mannose is recognized by the stem domain of another subunit. Structure-based functional studies confirmed that the dimeric structure is essential for FKRP enzymatic activity.


Assuntos
Distrofias Musculares/metabolismo , Açúcares de Nucleosídeo Difosfato/química , Açúcares de Nucleosídeo Difosfato/metabolismo , Pentosiltransferases/química , Pentosiltransferases/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Glicopeptídeos , Células HEK293 , Humanos , Modelos Moleculares , Distrofias Musculares/genética , Pentosiltransferases/genética , Fosfatos/metabolismo , Polissacarídeos/metabolismo , Conformação Proteica , Domínios Proteicos , Ribitol/metabolismo
7.
Biochim Biophys Acta Proteins Proteom ; 1868(2): 140304, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31689547

RESUMO

Nucleoside phosphorylases catalyze the reversible phosphorolysis of pyrimidine and purine nucleosides in the presence of phosphate. They are valuable catalysts in the synthesis of nucleosides and their analogues, which are often used as pharmaceuticals or their precursors. Thermostable nucleoside phosphorylases are promising biocatalysts, as they withstand harsh reaction conditions such as high pH or the addition of organic solvents. In this review, the characteristics and properties of thermostable nucleoside phosphorylases are described. Differences in amino acid content and protein structure were compared to their mesophilic homologues to identify features involved in thermostability. Substrate spectra of thermostable nucleoside phosphorylases were analyzed, and it is shown that thermostable nucleoside phosphorylases have a wider substrate spectrum than their mesophilic counterparts. Thus, thermostable nucleoside phosphorylases are interesting biocatalysts for industrial applications.


Assuntos
Pentosiltransferases/metabolismo , Archaea/enzimologia , Bactérias/enzimologia , Concentração de Íons de Hidrogênio , Cinética , Compostos Orgânicos/química , Pentosiltransferases/química , Estabilidade Proteica , Especificidade por Substrato , Temperatura
8.
Biochim Biophys Acta Proteins Proteom ; 1868(1): 140292, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676450

RESUMO

Enzymatic transglycosylation, a transfer of the carbohydrate moiety from one heterocyclic base to another, is catalyzed by nucleoside phosphorylases (NPs) and is being actively developed and applied for the synthesis of biologically important nucleosides. Here, we report an efficient one-step synthesis of 5-substitited pyrimidine ribonucleosides starting from 7-methylguanosine hydroiodide in the presence of nucleoside phosphorylases (NPs).


Assuntos
Proteínas de Bactérias/química , Escherichia coli/enzimologia , Pentosiltransferases/química , Ribonucleosídeos/química , Uridina/química , Proteínas de Bactérias/genética , Catálise , Glicosilação , Pentosiltransferases/genética , Proteínas Recombinantes/química
9.
Biochim Biophys Acta Proteins Proteom ; 1868(1): 140251, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31299354

RESUMO

Nowadays enzymatic synthesis of nucleic acid derivatives is gaining momentum over traditional chemical synthetic processes. Biotransformations catalyzed by whole cells or enzymes offer an ecofriendly and efficient alternative to the traditional multistep chemical methods, avoiding the use of chemical reagents and organic solvents that are expensive and environmentally harmful. Herein we report for the first time the covalent immobilization a uracil phosphoribosyltransferase (UPRT). In this sense, UPRT from Thermus thermophilus HB8 was immobilized onto glutaraldehyde-activated MagReSyn®Amine magnetic iron oxide porous microparticles (MTtUPRT). According to the catalyst load experiments, MTtUPRT3 was selected as optimal biocatalyst for further studies. MTtUPRT3 was active and stable in a broad range of temperature (70-100 °C) and in the pH interval 6-8, displaying maximum activity at 100 °C and pH 7 (activity 968 IU/gsupport, retained activity 100%). In addition, MTtUPRT3 could be reused up to 8 times in the synthesis of uridine-5'-monophosphate (UMP). Finally, MTtUPRT3 was successfully applied in the sustainable synthesis of different 5-modified uridine-5'-monophosphates at short times. Taking into account these results, MTtUPRT3 would emerge as a valuable biocatalyst for the synthesis of nucleoside monophosphates through an efficient and environmentally friendly methodology.


Assuntos
Enzimas Imobilizadas/metabolismo , Pentosiltransferases/metabolismo , Thermus thermophilus/enzimologia , Uridina Monofosfato/análogos & derivados , Uridina Monofosfato/biossíntese , Biocatálise , Compostos Férricos , Glutaral , Microesferas
10.
Genes (Basel) ; 10(11)2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31671740

RESUMO

The complete mutational spectrum of dystrophinopathies and limb-girdle muscular dystrophy (LGMD) remains unknown in Mexican population. Seventy-two unrelated Mexican male patients (73% of pediatric age) with clinical suspicion of muscular dystrophy and no evidence of DMD gene deletion on multiplex polymerase chain reaction (mPCR) analysis were analyzed by multiplex ligation-dependent probe amplification (MLPA). Those with a normal result were subjected to Sanger sequencing or to next-generation sequencing for DMD plus 10 selected LGMD-related genes. We achieved a diagnostic genotype in 80.5% (n = 58/72) of patients with predominance of dystrophinopathy-linked genotypes (68%, n = 49/72), followed by autosomal recessive LGMD-related genotypes (types 2A-R1, 2C-R5, 2E-R4, 2D-R3 and 2I-R9; 12.5%, n = 9/72). MLPA showed 4.2% of false-negatives for DMD deletions assessed by mPCR. Among the small DMD variants, 96.5% (n = 28/29) corresponded to null-alleles, most of which (72%) were inherited through a carrier mother. The FKRP p.[Leu276Ile]; [Asn463Asp] genotype is reported for the first time in Mexican patients as being associated with dilated cardiomyopathy. Absence of dysferlinopathies could be related to the small sample size and/or the predominantly pediatric age of patients. The employed strategy seems to be an affordable diagnosis approach for Mexican muscular dystrophy male patients and their families.


Assuntos
Distrofina/genética , Testes Genéticos/normas , Distrofia Muscular de Duchenne/genética , Mutação , Adolescente , Adulto , Criança , Pré-Escolar , Reações Falso-Negativas , Testes Genéticos/métodos , Genótipo , Humanos , Masculino , México , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/normas , Distrofia Muscular de Duchenne/patologia , Pentosiltransferases/genética
11.
Mutat Res ; 847: 503095, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31699345

RESUMO

I first became acquainted with the Ames test at the very beginning of my career in 1978, when my task at the National Institute of Health Sciences (Tokyo) was to screen for mutagenicity of food additives used in Japan, using the Ames test. I also used this test to research the metabolic activation mechanisms of chemical carcinogens, in particular, the analgesic drug, phenacetin. This chemical was not mutagenic in Salmonella typhimurium TA100 with standard 9000 × g supernatant of liver homogenates (S9) from rat but was mutagenic with hamster S9. It was revealed that hamster S9 had much higher deacetylation activities than rat S9, which accounts for the species difference. Then, my work was focused on molecular biology. We cloned the genes encoding nitroreductase and acetyltransferase in Salmonella typhimurium TA1538. Plasmids carrying these genes made strain TA98 more sensitive to mutagenic nitroarenes and aromatic amines. Because of their high sensitivity, the resulting strains such as YG1021 and YG1024 are widely used to monitor mutagenic nitroarenes and aromatic amines in complex mixtures. Later, we disrupted the genes encoding DNA polymerases in TA1538 and classified chemical mutagens into four classes depending on their use of different DNA polymerases. I was also involved in the generation of gpt delta transgenic rodent gene mutation assays, which examine the results of the Ames test in vivo. I have unintentionally developed my career under the influence of Dr. Ames and I would like to acknowledge his remarkable achievements in the field of environmental mutagenesis and carcinogenesis.


Assuntos
Testes de Mutagenicidade , Salmonella typhimurium/efeitos dos fármacos , Ativação Metabólica , Animais , Animais Geneticamente Modificados , Animais Endogâmicos , Proteínas de Bactérias/metabolismo , Boston , Carcinógenos/farmacocinética , Carcinógenos/toxicidade , Clonagem Molecular , Cricetinae , DNA Polimerase Dirigida por DNA/metabolismo , Exposição Ambiental/legislação & jurisprudência , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Feminino , Aditivos Alimentares/farmacocinética , Aditivos Alimentares/toxicidade , Japão , Camundongos , Microssomos Hepáticos/metabolismo , Mutagênicos/farmacocinética , Mutagênicos/toxicidade , Pentosiltransferases/genética , Ratos , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/classificação , Salmonella typhimurium/enzimologia , Salmonella typhimurium/genética
12.
Mutat Res ; 847: 403039, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31699347

RESUMO

Chemical safety evaluations require assessment of genetic toxicity. Transgenic rodent (TGR) assays permit enumeration of mutations in chromosomally-integrated targets contained in shuttle vectors. In order to improve in vitro mutagenicity assessment, and to substantially reduce animal use, in vitro assays using transgenic reporters have been developed. These assays are based on cells derived from TGRs, or cells transfected with transgenic shuttle vectors containing a mutation target. As part of the 7th International Workshop on Genotoxicity Testing, an In Vitro Mammalian Cell Gene Mutation Assay working group reviewed all published information pertaining to in vitro transgene mutagenicity assays; the utility, advantages and disadvantages of the assays were evaluated and discussed. The review revealed that over 20 TGR-based in vitro assays have been used to assess the mutagenic activity of over 150 agents. Overall, the Working Group considered in vitro transgene mutagenicity assays pragmatic tools for the safety evaluation of new and existing substances. A formal SWOT (strengths, weaknesses, opportunities, threats) analysis revealed advantages including the use of established scoring protocols, avoidance of laborious clone isolation and enumeration, ability to use metabolically competent primary cells, ability to detect different types of genetic damage, large dynamic range, and complementarity to in vivo TGR endpoints. Disadvantages include lack of validation and little consistency in protocols, the use of specialised reagents, the time and effort required for mutant enumeration, the use of some cell lines that lack metabolic capacity, and the need for multiple assays to cover all mutational mechanisms. Several assays have been partially validated, indicating promising reliability, reproducibility and applicability domain. Once in vitro transgene mutagenicity assays have been more thoroughly validated, they are well placed to augment or replace existing in vitro mammalian cell mutagenicity assays, particularly in cases where the in vivo TGR mutation assay is intended for follow-up.


Assuntos
Animais Geneticamente Modificados , Genes Reporter/efeitos dos fármacos , Testes de Mutagenicidade/métodos , Transgenes/efeitos dos fármacos , Animais , Biotransformação , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Proteínas de Escherichia coli/genética , Vetores Genéticos/genética , Humanos , Técnicas In Vitro , Óperon Lac , Pentosiltransferases/genética , Reprodutibilidade dos Testes , Projetos de Pesquisa , Roedores , Estudos de Validação como Assunto
14.
EMBO Rep ; 20(11): e47967, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31566294

RESUMO

Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α-dystroglycan-laminin interaction due to defective glycosylation of α-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of α-dystroglycan in iPSC-derived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of α-dystroglycan. Using human FKRP-iPSC-derived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of α-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Distroglicanas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Sequência de Bases , Sistemas CRISPR-Cas , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos/métodos , Distroglicanas/genética , Edição de Genes , Marcação de Genes , Loci Gênicos , Glicosilação/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imagem Molecular , Distrofias Musculares/tratamento farmacológico , Distrofias Musculares/etiologia , Distrofias Musculares/metabolismo , Mutação , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Pentosiltransferases/genética , Pentosiltransferases/metabolismo
15.
Eur J Med Chem ; 183: 111667, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31536893

RESUMO

Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is a recognized target for antimalarial chemotherapeutics. It synthesises all of the 6-oxopurine nucleoside monophosphates, IMP, GMP and XMP needed by the malarial parasite, Plasmodium falciparum (Pf). PfHGXPRT is also indirectly responsible for the synthesis of the adenosine monophosphate, AMP. The acyclic nucleoside phosphonates (ANPs) are a class of PfHGXPRT inhibitors. Prodrugs of these compounds are able to arrest the growth of Pf in cell culture. In the search for new inhibitors of PfHGXPRT, a series of sulfur containing ANPs (thia-ANPs) has been designed and synthesized. These compounds are based on the structure of 2-(phosphonoethoxy)ethylguanine (PEEG) and PEEHx which consist of a purine base (i.e. guanine or hypoxanthine) linked to a phosphonate group by five atoms i.e. four carbons and one oxygen. Here, PEEG and PEEHx were modified by substituting a sulfide, sulfoxide or a sulfone bridge for the oxygen atom in the linker. The effect of these substitutions on the Ki values for human HGPRT and PfHGXPRT was investigated and showed that most of the thia-ANPs distinctively favour PfHGXPRT. For example, the thia-analogue of PEEHx has a Ki value of 0.2 µM for PfHGXPRT, a value 25-fold lower than for the human counterpart. Prodrugs of these compounds have IC50 values in the 4-6 µM range in antimalarial cell-based assays, making them attractive compounds for further development as antimalarial drug leads.


Assuntos
Antimaláricos/síntese química , Nucleosídeos/síntese química , Organofosfonatos/síntese química , Pentosiltransferases/antagonistas & inibidores , Plasmodium falciparum/enzimologia , Sulfetos/química , Sulfonas/química , Sulfóxidos/química , Antimaláricos/farmacologia , Humanos , Estrutura Molecular , Nucleosídeos/farmacologia , Organofosfonatos/farmacologia , Oxirredução , Plasmodium falciparum/efeitos dos fármacos , Pró-Fármacos/síntese química , Pró-Fármacos/farmacologia , Relação Estrutura-Atividade , Termodinâmica
16.
Microbiology ; 165(11): 1198-1202, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31517594

RESUMO

Following penetration, the devastating rice blast fungus Magnaporthe oryzae, like some other important eukaryotic phytopathogens, grows in intimate contact with living plant cells before causing disease. Cell-to-cell growth during this biotrophic growth stage must involve nutrient acquisition, but experimental evidence for the internalization and metabolism of host-derived compounds is exceedingly sparse. This striking gap in our knowledge of the infection process undermines accurate conceptualization of the plant-fungal interaction. Here, through our general interest in Magnaporthe metabolism and with a specific focus on the signalling and redox cofactor nicotinamide adenine dinucleotide (NAD), we deleted the M. oryzae QPT1 gene encoding quinolinate phosphoribosyltransferase, catalyst of the last step in de novo NAD biosynthesis from tryptophan. We show how QPT1 is essential for axenic growth on minimal media lacking nicotinic acid (NA, an importable NAD precursor). However, Δqpt1 mutant strains were fully pathogenic, indicating de novo NAD biosynthesis is dispensable for lesion expansion following invasive hyphal growth in leaf tissue. Because overcoming the loss of de novo NAD biosynthesis in planta can only occur if importable NAD precursors (which solely comprise the NA, nicotinamide and nicotinamide riboside forms of vitamin B3) are accessible, we unexpectedly but unequivocally demonstrate that vitamin B3 can be acquired from the host and assimilated into Magnaporthe metabolism during growth in rice cells. Our results furnish a rare, experimentally determined example of host nutrient acquisition by a fungal plant pathogen and are significant in expanding our knowledge of events at the plant-fungus metabolic interface.


Assuntos
Magnaporthe/fisiologia , Niacinamida/metabolismo , Oryza/microbiologia , Doenças das Plantas/microbiologia , Meios de Cultura/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Magnaporthe/genética , Magnaporthe/metabolismo , Mutação , NAD/metabolismo , Niacina/metabolismo , Niacinamida/análise , Oryza/química , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Folhas de Planta/química , Folhas de Planta/microbiologia
17.
Nat Commun ; 10(1): 4116, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31511522

RESUMO

Damage-associated molecular patterns (DAMPs) are molecules that can be actively or passively released by injured tissues and that activate the immune system. Here we show that nicotinate phosphoribosyltransferase (NAPRT), detected by antibody-mediated assays and mass spectrometry, is an extracellular ligand for Toll-like receptor 4 (TLR4) and a critical mediator of inflammation, acting as a DAMP. Exposure of human and mouse macrophages to NAPRT activates the inflammasome and NF-κB for secretion of inflammatory cytokines. Furthermore, NAPRT enhances monocyte differentiation into macrophages by inducing macrophage colony-stimulating factor. These NAPRT-induced effects are independent of NAD-biosynthetic activity, but rely on NAPRT binding to TLR4. In line with our finding that NAPRT mediates endotoxin tolerance in vitro and in vivo, sera from patients with sepsis contain the highest levels of NAPRT, compared to patients with other chronic inflammatory conditions. Together, these data identify NAPRT as a endogenous ligand for TLR4 and a mediator of inflammation.


Assuntos
Espaço Extracelular/metabolismo , Inflamação/enzimologia , Pentosiltransferases/metabolismo , Receptor 4 Toll-Like/metabolismo , Diferenciação Celular , Líquido Extracelular/enzimologia , Humanos , Inflamação/genética , Inflamação/patologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/metabolismo , Monócitos/citologia , Células Mieloides/metabolismo , Nicotinamida Fosforribosiltransferase/química , Nicotinamida Fosforribosiltransferase/metabolismo , Pentosiltransferases/sangue , Pentosiltransferases/química , Ligação Proteica , Fatores de Risco , Sepse/sangue , Sepse/enzimologia
18.
Proc Natl Acad Sci U S A ; 116(38): 19126-19135, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31481610

RESUMO

Queuosine (Q) is a complex tRNA modification widespread in eukaryotes and bacteria that contributes to the efficiency and accuracy of protein synthesis. Eukaryotes are not capable of Q synthesis and rely on salvage of the queuine base (q) as a Q precursor. While many bacteria are capable of Q de novo synthesis, salvage of the prokaryotic Q precursors preQ0 and preQ1 also occurs. With the exception of Escherichia coli YhhQ, shown to transport preQ0 and preQ1, the enzymes and transporters involved in Q salvage and recycling have not been well described. We discovered and characterized 2 Q salvage pathways present in many pathogenic and commensal bacteria. The first, found in the intracellular pathogen Chlamydia trachomatis, uses YhhQ and tRNA guanine transglycosylase (TGT) homologs that have changed substrate specificities to directly salvage q, mimicking the eukaryotic pathway. The second, found in bacteria from the gut flora such as Clostridioides difficile, salvages preQ1 from q through an unprecedented reaction catalyzed by a newly defined subgroup of the radical-SAM enzyme family. The source of q can be external through transport by members of the energy-coupling factor (ECF) family or internal through hydrolysis of Q by a dedicated nucleosidase. This work reinforces the concept that hosts and members of their associated microbiota compete for the salvage of Q precursors micronutrients.


Assuntos
Proteínas de Bactérias/metabolismo , Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/metabolismo , Infecções por Clostridium/metabolismo , Clostridium difficile/metabolismo , Guanina/análogos & derivados , Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/crescimento & desenvolvimento , Infecções por Clostridium/microbiologia , Clostridium difficile/crescimento & desenvolvimento , Guanina/metabolismo , Humanos , Pentosiltransferases/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Transdução de Sinais , Especificidade por Substrato
20.
Org Biomol Chem ; 17(34): 7891-7899, 2019 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-31397456

RESUMO

Insight into the catalytic mechanism of Lactobacillus leichmannii nucleoside 2'-deoxyribosyltransferase (LlNDT) has been gained by calculating a quantum mechanics-molecular mechanics (QM/MM) free-energy landscape of the reaction within the enzyme active site. Our results support an oxocarbenium species as the reaction intermediate and thus an SN1 reaction mechanism in this family of bacterial enzymes. Our mechanistic proposal is validated by comparing experimental kinetic data on the impact of the single amino acid replacements Tyr7, Glu98 and Met125 with Ala, Asp and Ala/norLeu, respectively, and accounts for the specificity shown by this enzyme on a non-natural substrate. This work broadens our understanding of enzymatic C-N bond cleavage and C-N bond formation.


Assuntos
Pentosiltransferases/química , Domínio Catalítico , Cinética , Lactobacillus leichmannii/enzimologia , Modelos Químicos , Simulação de Dinâmica Molecular , Estudo de Prova de Conceito , Conformação Proteica , Teoria Quântica , Termodinâmica
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