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1.
Comput Biol Chem ; 83: 107150, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31733620

RESUMO

Nicotinamidase is a key enzyme for the salvage pathway catalyzing the first step for the conversion of nicotinamide (NAm) to nicotinic acid (NA) required for the synthesis of Nicotinamide Adenine Dinucleotide (NAD+) in the subsequent steps. Leishmania protozoan parasites are NAD+ auxotrophs and need precursors (nicotinamide, nicotinic acid, nicotinamide riboside) from their host environment to synthesize NAD+ for their survival. Interestingly, absence of this enzyme in higher eukaryotes and its absolute requirement in the developmental cycle of Leishmania has led nicotinamidase an attractive drug target towards leishmaniasis. Hence, we report some potential inhibitors for nicotinamidase screened based on 3-D pharmacophore model consisting of "ML", "Hyd|Aro", "Acc" and "Excl vol" features. Subsequently, dynamics simulation studies validate the proposed pharmacophore model suggesting its reliability for future studies. Furthermore, these essential site-specific features will help in enhancing the inhibition of nicotinamidase activity. Results of our study suggest that blocking of active site of nicotinamidase by the identified lead inhibitor will have major impact on the infectious processes and the survival of the parasite. Furthermore, due to the structural homology in the enzyme among L. donovani, L. infantum, L. major, we anticipate that our study would help to design more potent drug candidates against leshmaniasis for these three species.


Assuntos
Simulação por Computador , Inibidores Enzimáticos/farmacologia , Leishmania donovani/enzimologia , Nicotinamidase/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Modelos Moleculares , Estrutura Molecular , Nicotinamidase/metabolismo , Filogenia
2.
Plant Physiol ; 179(4): 1810-1821, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30692220

RESUMO

DNA methylation plays an important role in diverse developmental processes in many eukaryotes, including the response to environmental stress. Abscisic acid (ABA) is a plant hormone that is up-regulated under stress. The involvement of DNA methylation in the ABA response has been reported but is poorly understood. DNA demethylation is a reverse process of DNA methylation and often induces structural changes of chromatin leading to transcriptional activation. In Arabidopsis (Arabidopsis thaliana), active DNA demethylation depends on the activity of REPRESSOR OF SILENCING 1 (ROS1), which directly excises 5-methylcytosine from DNA. Here we showed that ros1 mutants were hypersensitive to ABA during early seedling development and root elongation. Expression levels of some ABA-inducible genes were decreased in ros1 mutants, and more than 60% of their proximal regions became hypermethylated, indicating that a subset of ABA-inducible genes are under the regulation of ROS1-dependent DNA demethylation. Notable among them is NICOTINAMIDASE 3 (NIC3) that encodes an enzyme that converts nicotinamide to nicotinic acid in the NAD+ salvage pathway. Many enzymes in this pathway are known to be involved in stress responses. The nic3 mutants display hypersensitivity to ABA, whereas overexpression of NIC3 restores normal ABA responses. Our data suggest that NIC3 is responsive to ABA but requires ROS1-mediated DNA demethylation at the promoter as a prerequisite to transcriptional activation. These findings suggest that ROS1-induced active DNA demethylation maintains the active state of NIC3 transcription in response to ABA.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Desmetilação do DNA , Proteínas Nucleares/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Metilação de DNA , Epigenômica , Regulação da Expressão Gênica de Plantas , Redes e Vias Metabólicas/genética , Nicotinamidase/genética , Nicotinamidase/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo
3.
Biochem Biophys Res Commun ; 503(4): 2906-2911, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30107912

RESUMO

The nicotinamidase/pyrazinamidase PncA is a member of a large family of hydrolase enzymes that catalyze the deamination of nicotinamide to nicotinic acid. PncA also functions as a pyrazinamidase in a wide variety of eubacteria and is an essential coenzyme in many cellular redox reactions in living systems. We report the crystal structure of substrate-free PncA from Bacillus subtilis (BsPncA) at 2.0 Šresolution to improve our understanding of the PncA family. The structure of BsPncA consists of an α/ß domain and a subdomain. The subdomain of BsPncA has a different conformation than that of PncA enzymes from other organisms. The B-factor analysis revealed a rigid structure of the α/ß domain, while the subdomain is highly flexible. Both dimers and tetramers were observed in BsPncA protein crystals, but only dimers were observed in solution. Our results provide useful information that will further enhance our understanding of the molecular functions of PncA family members.


Assuntos
Amidoidrolases/química , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Cristalografia por Raios X , Nicotinamidase , Conformação Proteica , Domínios Proteicos , Multimerização Proteica
4.
J Bacteriol ; 200(11)2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29555696

RESUMO

Many organisms possess pathways that regenerate NAD+ from its degradation products, and two pathways are known to salvage NAD+ from nicotinamide (Nm). One is a four-step pathway that proceeds through deamination of Nm to nicotinic acid (Na) by Nm deamidase and phosphoribosylation to nicotinic acid mononucleotide (NaMN), followed by adenylylation and amidation. Another is a two-step pathway that does not involve deamination and directly proceeds with the phosphoribosylation of Nm to nicotinamide mononucleotide (NMN), followed by adenylylation. Judging from genome sequence data, the hyperthermophilic archaeon Thermococcus kodakarensis is supposed to utilize the four-step pathway, but the fact that the adenylyltransferase encoded by TK0067 recognizes both NMN and NaMN also raises the possibility of a two-step salvage mechanism. Here, we examined the substrate specificity of the recombinant TK1676 protein, annotated as nicotinic acid phosphoribosyltransferase. The TK1676 protein displayed significant activity toward Na and phosphoribosyl pyrophosphate (PRPP) and only trace activity with Nm and PRPP. We further performed genetic analyses on TK0218 (quinolinic acid phosphoribosyltransferase) and TK1650 (Nm deamidase), involved in de novo biosynthesis and four-step salvage of NAD+, respectively. The ΔTK0218 mutant cells displayed growth defects in a minimal synthetic medium, but growth was fully restored with the addition of Na or Nm. The ΔTK0218 ΔTK1650 mutant cells did not display growth in the minimal medium, and growth was restored with the addition of Na but not Nm. The enzymatic and genetic analyses strongly suggest that NAD+ salvage in T. kodakarensis requires deamination of Nm and proceeds through the four-step pathway.IMPORTANCE Hyperthermophiles must constantly deal with increased degradation rates of their biomolecules due to their high growth temperatures. Here, we identified the pathway that regenerates NAD+ from nicotinamide (Nm) in the hyperthermophilic archaeon Thermococcus kodakarensis The organism utilizes a four-step pathway that initially hydrolyzes the amide bond of Nm to generate nicotinic acid (Na), followed by phosphoribosylation, adenylylation, and amidation. Although the two-step pathway, consisting of only phosphoribosylation of Nm and adenylylation, seems to be more efficient, Nm mononucleotide in the two-step pathway is much more thermolabile than Na mononucleotide, the corresponding intermediate in the four-step pathway. Although NAD+ itself is thermolabile, this may represent an example of a metabolism that has evolved to avoid the use of thermolabile intermediates.


Assuntos
NAD/metabolismo , Nicotinamidase/metabolismo , Nucleotidiltransferases/metabolismo , Pentosiltransferases/metabolismo , Thermococcus/metabolismo , Desaminação , Temperatura Alta , Niacinamida/metabolismo , Nicotinamidase/genética , Mononucleotídeo de Nicotinamida/análogos & derivados , Mononucleotídeo de Nicotinamida/metabolismo , Ácidos Nicotínicos/metabolismo , Nucleotidiltransferases/genética , Pentosiltransferases/genética , Proteínas Recombinantes , Especificidade por Substrato , Thermococcus/genética , Thermococcus/crescimento & desenvolvimento
5.
PLoS One ; 12(7): e0181561, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28750065

RESUMO

Nicotinamidases are amidohydrolases that convert nicotinamide into nicotinic acid, contributing to NAD+ homeostasis in most organisms. In order to increase the number of nicotinamidases described to date, this manuscript characterizes a nicotinamidase obtained from a metagenomic library fosmid clone (JFF054_F02) obtained from a geothermal water stream microbial mat community in a Japanese epithermal mine. The enzyme showed an optimum temperature of 90°C, making it the first hyperthermophilic bacterial nicotinamidase to be characterized, since the phylogenetic analysis of this fosmid clone placed it in a clade of uncultured geothermal bacteria. The enzyme, named as UbNic, not only showed an alkaline optimum pH, but also a biphasic pH dependence of its kcat, with a maximum at pH 9.5-10.0. The two pKa values obtained were 4.2 and 8.6 for pKes1 and pKes2, respectively. These results suggest a possible flexible catalytic mechanism for nicotinamidases, which reconciles the two previously proposed mechanisms. In addition, the enzyme showed a high catalytic efficiency, not only toward nicotinamide, but also toward other nicotinamide analogs. Its mutational analysis showed that a tryptophan (W83) is needed in one of the faces of the active site to maintain low Km values toward all the substrates tested. Furthermore, UbNic proved to contain a Fe2+ ion in its metal binding site, and was revealed to belong to a new nicotinamidase subgroup. All these characteristics, together with its high pH- and thermal stability, distinguish UbNic from previously described nicotinamidases, and suggest that a wide diversity of enzymes remains to be discovered in extreme environments.


Assuntos
Bactérias/enzimologia , Fontes Termais/microbiologia , Microbiota , Nicotinamidase/metabolismo , Rios/microbiologia , Microbiologia da Água , Aldeídos/metabolismo , Sequência de Aminoácidos , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Proteínas Mutantes/metabolismo , Nicotinamidase/antagonistas & inibidores , Nicotinamidase/química , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Temperatura
6.
J Bacteriol ; 199(17)2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28630126

RESUMO

NAD (NAD+) is a cofactor related to many cellular processes. This cofactor is known to be unstable, especially at high temperatures, where it chemically decomposes to nicotinamide and ADP-ribose. Bacteria, yeast, and higher organisms possess the salvage pathway for reconstructing NAD+ from these decomposition products; however, the importance of the salvage pathway for survival is not well elucidated, except for in pathogens lacking the NAD+de novo synthesis pathway. Herein, we report the importance of the NAD+ salvage pathway in the thermophilic bacterium Thermus thermophilus HB8 at high temperatures. We identified the gene encoding nicotinamidase (TTHA0328), which catalyzes the first reaction of the NAD+ salvage pathway. This recombinant enzyme has a high catalytic activity against nicotinamide (Km of 17 µM, kcat of 50 s-1, kcat/Km of 3.0 × 103 s-1 · mM-1). Deletion of this gene abolished nicotinamide deamination activity in crude extracts of T. thermophilus and disrupted the NAD+ salvage pathway in T. thermophilus Disruption of the salvage pathway led to the severe growth retardation at a higher temperature (80°C), owing to the drastic decrease in the intracellular concentrations of NAD+ and NADH.IMPORTANCE NAD+ and other nicotinamide cofactors are essential for cell metabolism. These molecules are unstable and decompose, even under the physiological conditions in most organisms. Thermophiles can survive at high temperatures where NAD+ decomposition is, in general, more rapid. This study emphasizes that NAD+ instability and its homeostasis can be one of the important factors for thermophile survival in extreme temperatures.


Assuntos
Deleção de Genes , NAD/metabolismo , Nicotinamidase/metabolismo , Thermus thermophilus/enzimologia , Thermus thermophilus/efeitos da radiação , Temperatura Alta , Cinética , Niacinamida/metabolismo , Nicotinamidase/genética , Thermus thermophilus/genética , Thermus thermophilus/crescimento & desenvolvimento
7.
EMBO Rep ; 17(12): 1829-1843, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27799288

RESUMO

Changes in histone modifications are an attractive model through which environmental signals, such as diet, could be integrated in the cell for regulating its lifespan. However, evidence linking dietary interventions with specific alterations in histone modifications that subsequently affect lifespan remains elusive. We show here that deletion of histone N-alpha-terminal acetyltransferase Nat4 and loss of its associated H4 N-terminal acetylation (N-acH4) extend yeast replicative lifespan. Notably, nat4Δ-induced longevity is epistatic to the effects of calorie restriction (CR). Consistent with this, (i) Nat4 expression is downregulated and the levels of N-acH4 within chromatin are reduced upon CR, (ii) constitutive expression of Nat4 and maintenance of N-acH4 levels reduces the extension of lifespan mediated by CR, and (iii) transcriptome analysis indicates that nat4Δ largely mimics the effects of CR, especially in the induction of stress-response genes. We further show that nicotinamidase Pnc1, which is typically upregulated under CR, is required for nat4Δ-mediated longevity. Collectively, these findings establish histone N-acH4 as a regulator of cellular lifespan that links CR to increased stress resistance and longevity.


Assuntos
Restrição Calórica , Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Acetiltransferase N-Terminal D/deficiência , Acetiltransferase N-Terminal D/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Acetilação , Cromatina/metabolismo , Regulação para Baixo , Perfilação da Expressão Gênica , Histona Acetiltransferases/metabolismo , Longevidade , Acetiltransferase N-Terminal D/genética , Nicotinamidase/genética , Nicotinamidase/metabolismo , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Ativação Transcricional
8.
Genetics ; 204(2): 569-579, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27527516

RESUMO

Nicotinamide is both a reaction product and an inhibitor of the conserved sirtuin family of deacetylases, which have been implicated in a broad range of cellular functions in eukaryotes from yeast to humans. Phenotypes observed following treatment with nicotinamide are most often assumed to stem from inhibition of one or more of these enzymes. Here, we used this small molecule to inhibit multiple sirtuins at once during treatment with DNA damaging agents in the Saccharomyces cerevisiae model system. Since sirtuins have been previously implicated in the DNA damage response, we were surprised to observe that nicotinamide actually increased the survival of yeast cells exposed to the DNA damage agent MMS. Remarkably, we found that enhanced resistance to MMS in the presence of nicotinamide was independent of all five yeast sirtuins. Enhanced resistance was also independent of the nicotinamide salvage pathway, which uses nicotinamide as a substrate to generate NAD+, and of a DNA damage-induced increase in the salvage enzyme Pnc1 Our data suggest a novel and unexpected function for nicotinamide that has broad implications for its use in the study of sirtuin biology across model systems.


Assuntos
Dano ao DNA/genética , Histona Desacetilases do Grupo III/genética , Nicotinamidase/genética , Proteínas de Saccharomyces cerevisiae/genética , Sirtuínas/genética , Dano ao DNA/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Histona Desacetilases do Grupo III/biossíntese , Metanossulfonato de Metila/toxicidade , Niacinamida/farmacologia , Fenótipo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Sirtuínas/biossíntese
9.
Appl Environ Microbiol ; 82(19): 5815-23, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27451449

RESUMO

UNLABELLED: Riemerella anatipestifer is a major bacterial pathogen that causes septicemic and exudative diseases in domestic ducks. In our previous study, we found that deletion of the AS87_01735 gene significantly decreased the bacterial virulence of R. anatipestifer strain Yb2 (mutant RA625). The AS87_01735 gene was predicted to encode a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD(+) salvage pathway. In this study, the AS87_01735 gene was expressed and identified as the PncA-encoding gene, using an enzymatic assay. Western blot analysis demonstrated that R. anatipestifer PncA was localized to the cytoplasm. The mutant strain RA625 (named Yb2ΔpncA in this study) showed a similar growth rate but decreased NAD(+) quantities in both the exponential and stationary phases in tryptic soy broth culture, compared with the wild-type strain Yb2. In addition, Yb2ΔpncA-infected ducks showed much lower bacterial loads in their blood, and no visible histological changes were observed in the heart, liver, and spleen. Furthermore, Yb2ΔpncA immunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Our results suggest that the R. anatipestifer AS87_01735 gene encodes PncA, which is an important virulence factor, and that the Yb2ΔpncA mutant can be used as a novel live vaccine candidate. IMPORTANCE: Riemerella anatipestifer is reported worldwide as a cause of septicemic and exudative diseases of domestic ducks. The pncA gene encodes a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD(+) salvage pathway. In this study, we identified and characterized the pncA-homologous gene AS87_01735 in R. anatipestifer strain Yb2. R. anatipestifer PncA is a cytoplasmic protein that possesses similar PncA activity, compared with other organisms. Generation of the pncA mutant Yb2ΔpncA led to a decrease in the NAD(+) content, which was associated with decreased capacity for invasion and attenuated virulence in ducks. Furthermore, Yb2ΔpncA immunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Altogether, these results suggest that PncA contributes to the virulence of R. anatipestifer and that the Yb2ΔpncA mutant can be used as a novel live vaccine candidate.


Assuntos
Proteínas de Bactérias/genética , Patos , Infecções por Flavobacteriaceae/veterinária , Nicotinamidase/genética , Doenças das Aves Domésticas/imunologia , Riemerella/genética , Fatores de Virulência/genética , Animais , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/microbiologia , Infecções por Flavobacteriaceae/imunologia , Infecções por Flavobacteriaceae/microbiologia , Expressão Gênica , Imunização/veterinária , Nicotinamidase/imunologia , Nicotinamidase/metabolismo , Doenças das Aves Domésticas/microbiologia , Riemerella/imunologia , Riemerella/metabolismo , Deleção de Sequência , Vacinas Atenuadas/imunologia , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismo
10.
Biochim Biophys Acta ; 1863(1): 148-56, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26516056

RESUMO

Saccharomyces cerevisiae glycerol phosphate dehydrogenase 1 (Gpd1) and nicotinamidase (Pnc1) are two stress-induced enzymes. Both enzymes are predominantly localised to peroxisomes at normal growth conditions, but were reported to localise to the cytosol and nucleus upon exposure of cells to stress. Import of both proteins into peroxisomes depends on the peroxisomal targeting signal 2 (PTS2) receptor Pex7. Gpd1 contains a PTS2, however, Pnc1 lacks this sequence. Here we show that Pnc1 physically interacts with Gpd1, which is required for piggy-back import of Pnc1 into peroxisomes. Quantitative fluorescence microscopy analyses revealed that the levels of both proteins increased in peroxisomes and in the cytosol upon exposure of cells to stress. However, upon exposure of cells to stress we also observed enhanced cytosolic levels of the control PTS2 protein thiolase, when produced under control of the GPD1 promoter. This suggests that these conditions cause a partial defect in PTS2 protein import, probably because the PTS2 import pathway is easily saturated.


Assuntos
Glicerol-3-Fosfato Desidrogenase (NAD+)/metabolismo , Nicotinamidase/metabolismo , Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/fisiologia , Glicerol-3-Fosfato Desidrogenase (NAD+)/genética , Nicotinamidase/genética , Receptor 2 de Sinal de Orientação para Peroxissomos , Peroxissomos/genética , Transporte Proteico/fisiologia , Receptores Citoplasmáticos e Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
11.
J Biol Chem ; 290(42): 25333-42, 2015 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-26276932

RESUMO

Proteins designated for peroxisomal protein import harbor one of two common peroxisomal targeting signals (PTS). In the yeast Saccharomyces cerevisiae, the oleate-induced PTS2-dependent import of the thiolase Fox3p into peroxisomes is conducted by the soluble import receptor Pex7p in cooperation with the auxiliary Pex18p, one of two supposedly redundant PTS2 co-receptors. Here, we report on a novel function for the co-receptor Pex21p, which cannot be fulfilled by Pex18p. The data establish Pex21p as a general co-receptor in PTS2-dependent protein import, whereas Pex18p is especially important for oleate-induced import of PTS2 proteins. The glycerol-producing PTS2 protein glycerol-3-phosphate dehydrogenase Gpd1p shows a tripartite localization in peroxisomes, in the cytosol, and in the nucleus under osmotic stress conditions. We show the following: (i) Pex21p is required for peroxisomal import of Gpd1p as well as a key enzyme of the NAD(+) salvage pathway, Pnc1p; (ii) Pnc1p, a nicotinamidase without functional PTS2, is co-imported into peroxisomes by piggyback transport via Gpd1p. Moreover, the specific transport of these two enzymes into peroxisomes suggests a novel regulatory role for peroxisomes under various stress conditions.


Assuntos
Proteínas de Transporte/fisiologia , Glicerol-3-Fosfato Desidrogenase (NAD+)/metabolismo , Nicotinamidase/metabolismo , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Dimerização , Transporte Proteico , Saccharomyces cerevisiae/fisiologia , Estresse Fisiológico
12.
Molecules ; 19(10): 15735-53, 2014 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-25268724

RESUMO

Nicotinamidase (Nic) is a key zinc-dependent enzyme in NAD metabolism that catalyzes the hydrolysis of nicotinamide to give nicotinic acid. A multi-scale computational approach has been used to investigate the catalytic mechanism, substrate binding and roles of active site residues of Nic from Streptococcus pneumoniae (SpNic). In particular, density functional theory (DFT), molecular dynamics (MD) and ONIOM quantum mechanics/molecular mechanics (QM/MM) methods have been employed. The overall mechanism occurs in two stages: (i) formation of a thioester enzyme-intermediate (IC2) and (ii) hydrolysis of the thioester bond to give the products. The polar protein environment has a significant effect in stabilizing reaction intermediates and in particular transition states. As a result, both stages effectively occur in one step with Stage 1, formation of IC2, being rate limiting barrier with a cost of 53.5 kJ·mol-1 with respect to the reactant complex, RC. The effects of dispersion interactions on the overall mechanism were also considered but were generally calculated to have less significant effects with the overall mechanism being unchanged. In addition, the active site lysyl (Lys103) is concluded to likely play a role in stabilizing the thiolate of Cys136 during the reaction.


Assuntos
Modelos Químicos , Modelos Moleculares , Nicotinamidase/química , Nicotinamidase/metabolismo , Streptococcus pneumoniae/enzimologia , Sítios de Ligação , Catálise , Domínio Catalítico , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
13.
PLoS One ; 9(9): e106496, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25188219

RESUMO

Saccharomyces cerevisiae is calorie-restricted by lowering glucose from 2% to 0.5%. Under low glucose conditions, replicative lifespan is extended in a manner that depends on the NAD+-dependent protein lysine deacetylase Sir2 and NAD+ salvage enzymes. Because NAD+ is required for glucose utilization and Sir2 function, it was postulated that glucose levels alter the levels of NAD+ metabolites that tune Sir2 function. Though NAD+ precursor vitamins, which increase the levels of all NAD+ metabolites, can extend yeast replicative lifespan, glucose restriction does not significantly change the levels or ratios of intracellular NAD+ metabolites. To test whether glucose restriction affects protein copy numbers, we developed a technology that combines the measurement of Urh1 specific activity and quantification of relative expression between Urh1 and any other protein. The technology was applied to obtain the protein copy numbers of enzymes involved in NAD+ metabolism in rich and synthetic yeast media. Our data indicated that Sir2 and Pnc1, two enzymes that sequentially convert NAD+ to nicotinamide and then to nicotinic acid, are up-regulated by glucose restriction in rich media, and that Pnc1 alone is up-regulated in synthetic media while levels of all other enzymes are unchanged. These data suggest that production or export of nicotinic acid might be a connection between NAD+ and calorie restriction-mediated lifespan extension in yeast.


Assuntos
Metaboloma , NAD/metabolismo , Saccharomyces cerevisiae/metabolismo , Niacina/metabolismo , Niacinamida/metabolismo , Nicotinamidase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/metabolismo
14.
Tuberculosis (Edinb) ; 94(6): 644-8, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25199451

RESUMO

Recombinant wild-pyrazinamidase from H37Rv Mycobacterium tuberculosis was analyzed by gel electrophoresis under differential reducing conditions to evaluate its quaternary structure. PZAse was fractionated by size exclusion chromatography under non-reducing conditions. PZAse activity was measured and mass spectrometry analysis was performed to determine the identity of proteins by de novo sequencing and to determine the presence of disulfide bonds. This study confirmed that M. tuberculosis wild type PZAse was able to form homo-dimers in vitro. Homo-dimers showed a slightly lower specific PZAse activity compared to monomeric PZAse. PZAse dimers were dissociated into monomers in response to reducing conditions. Mass spectrometry analysis confirmed the existence of disulfide bonds (C72-C138 and C138-C138) stabilizing the quaternary structure of the PZAse homo-dimer.


Assuntos
Amidoidrolases/metabolismo , Mycobacterium tuberculosis/enzimologia , Nicotinamidase/metabolismo , Sítios de Ligação , Cromatografia em Gel/métodos , Dissulfetos/metabolismo , Humanos , Espectrometria de Massas/métodos , Modelos Moleculares , Mycobacterium tuberculosis/metabolismo
15.
DNA Repair (Amst) ; 23: 49-58, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25096760

RESUMO

Pyridine nucleotides are essential coenzymes in many cellular redox reactions in all living systems. In addition to functioning as a redox carrier, NAD(+) is also a required co-substrate for the conserved sirtuin deacetylases. Sirtuins regulate transcription, genome maintenance and metabolism and function as molecular links between cells and their environment. Maintaining NAD(+) homeostasis is essential for proper cellular function and aberrant NAD(+) metabolism has been implicated in a number of metabolic- and age-associated diseases. Recently, NAD(+) metabolism has been linked to the phosphate-responsive signaling pathway (PHO pathway) in the budding yeast Saccharomyces cerevisiae. Activation of the PHO pathway is associated with the production and mobilization of the NAD(+) metabolite nicotinamide riboside (NR), which is mediated in part by PHO-regulated nucleotidases. Cross-regulation between NAD(+) metabolism and the PHO pathway has also been reported; however, detailed mechanisms remain to be elucidated. The PHO pathway also appears to modulate the activities of common downstream effectors of multiple nutrient-sensing pathways (Ras-PKA, TOR, Sch9/AKT). These signaling pathways were suggested to play a role in calorie restriction-mediated beneficial effects, which have also been linked to Sir2 function and NAD(+) metabolism. Here, we discuss the interactions of these pathways and their potential roles in regulating NAD(+) metabolism. In eukaryotic cells, intracellular compartmentalization facilitates the regulation of enzymatic functions and also concentrates or sequesters specific metabolites. Various NAD(+)-mediated cellular functions such as mitochondrial oxidative phosphorylation are compartmentalized. Therefore, we also discuss several key players functioning in mitochondrial, cytosolic and vacuolar compartmentalization of NAD(+) intermediates, and their potential roles in NAD(+) homeostasis. To date, it remains unclear how NAD(+) and NAD(+) intermediates shuttle between different cellular compartments. Together, these studies provide a molecular basis for how NAD(+) homeostasis factors and the interacting signaling pathways confer metabolic flexibility and contribute to maintaining cell fitness and genome stability.


Assuntos
NAD/metabolismo , Saccharomyces cerevisiae/metabolismo , Compartimento Celular , Citosol/metabolismo , Redes e Vias Metabólicas , Mitocôndrias/metabolismo , NAD/biossíntese , Nicotinamidase/metabolismo , Fosfatos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/metabolismo , Vacúolos/metabolismo
16.
Nucleic Acids Res ; 42(13): 8486-99, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24981510

RESUMO

In Saccharomyces cerevisiae, the stability of highly repetitive rDNA array is maintained through transcriptional silencing. Recently, a ß-1,3-glucanosyltransferase Gas1 has been shown to play a significant role in the regulation of transcriptional silencing in S. cerevisiae. Here, we show that the gas1Δ mutation increases rDNA silencing in a Sir2-dependent manner. Remarkably, the gas1Δ mutation induces nuclear localization of Msn2/4 and stimulates the expression of PNC1, a gene encoding a nicotinamidase that functions as a Sir2 activator. The lack of enzymatic activity of Gas1 or treatment with a cell wall-damaging agent, Congo red, exhibits effects similar to those of the gas1Δ mutation. Furthermore, the loss of Gas1 or Congo red treatment lowers the cAMP-dependent protein kinase (PKA) activity in a cell wall integrity MAP kinase Slt2-dependent manner. Collectively, our results suggest that the dysfunction of Gas1 plays a positive role in the maintenance of rDNA integrity by decreasing PKA activity and inducing the accumulation of Msn2/4 in the nucleus. It seems that nuclear-localized Msn2/4 stimulate the expression of Pnc1, thereby enhancing the association of Sir2 with rDNA and promoting rDNA stability.


Assuntos
DNA Ribossômico , Regulação Fúngica da Expressão Gênica , Inativação Gênica , Glucana Endo-1,3-beta-D-Glucosidase/fisiologia , Glicoproteínas de Membrana/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/metabolismo , Vermelho Congo , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Glucana Endo-1,3-beta-D-Glucosidase/genética , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Nicotinamidase/biossíntese , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo
17.
Plant Biol (Stuttg) ; 16(6): 1140-4, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24802616

RESUMO

Methyl jasmonate (MeJA) signalling shares several signal components with abscisic acid (ABA) signalling in guard cells. Cyclic adenosine 5'-diphosphoribose (cADPR) and cyclic guanosine 3',5'-monophosphate (cGMP) are second messengers in ABA-induced stomatal closure. In order to clarify involvement of cADPR and cGMP in MeJA-induced stomatal closure in Arabidopsis thaliana (Col-0), we investigated effects of an inhibitor of cADPR synthesis, nicotinamide (NA), and an inhibitor of cGMP synthesis, LY83583 (LY, 6-anilino-5,8-quinolinedione), on MeJA-induced stomatal closure. Treatment with NA and LY inhibited MeJA-induced stomatal closure. NA inhibited MeJA-induced reactive oxygen species (ROS) accumulation and nitric oxide (NO) production in guard cells. NA and LY suppressed transient elevations elicited by MeJA in cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) in guard cells. These results suggest that cADPR and cGMP positively function in [Ca(2+)]cyt elevation in MeJA-induced stomatal closure, are signalling components shared with ABA-induced stomatal closure in Arabidopsis, and that cADPR is required for MeJA-induced ROS accumulation and NO production in Arabidopsis guard cells.


Assuntos
Acetatos/farmacologia , Cálcio/metabolismo , ADP-Ribose Cíclica/metabolismo , GMP Cíclico/metabolismo , Ciclopentanos/farmacologia , Oxilipinas/farmacologia , Estômatos de Plantas/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Aminoquinolinas , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Nicotinamidase , Óxido Nítrico/metabolismo , Folhas de Planta/citologia , Estômatos de Plantas/metabolismo
18.
Biochemistry (Mosc) ; 79(1): 54-61, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24512664

RESUMO

Nicotinamidase is involved in the maintenance of NAD+ homeostasis and in the NAD+ salvage pathway of most prokaryotes, and it is considered as a possible drug target. The gene (ASAC_0847) encoding a hypothetical nicotinamidase has been found in the genome of the thermophilic archaeon Acidilobus saccharovorans. The product of this gene, NA_As0847, has been expressed in Escherichia coli, isolated, and characterized as a Fe(2+)-containing nicotinamidase (k(cat)/K(m) = 427 mM(-1)·sec(-1))/pyrazinamidase (k(cat)/K(m) = 331 mM(-1)·sec(-1)). NA_As0847 is a homodimer with molecular mass 46.4 kDa. The enzyme has high thermostability (T(1/2) (60°C) = 180 min, T(1/2) (80°C) = 35 min) and thermophilicity (T(opt) = 90°C, E(a) = 30.2 ± 1.0 kJ/mol) and broad pH interval of activity, with the optimum at pH 7.5. Special features of NA_As0847 are the presence of Fe2+ instead of Zn2+ in the active site of the enzyme and inhibition of the enzyme activity by Zn2+ at micromolar concentrations. Analysis of the amino acid sequence revealed a new motif of the metal-binding site (DXHXXXDXXEXXXWXXH) for homological archaeal nicotinamidases.


Assuntos
Proteínas Arqueais/metabolismo , Crenarchaeota/enzimologia , Nicotinamidase/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Domínio Catalítico , Crenarchaeota/genética , Dimerização , Escherichia coli/metabolismo , Genoma Arqueal , Íons/química , Cinética , Dados de Sequência Molecular , Nicotinamidase/química , Nicotinamidase/genética , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Temperatura , Zinco/química , Zinco/metabolismo
19.
Org Biomol Chem ; 12(8): 1265-77, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24413890

RESUMO

Nicotinamidase (Pnc1) is a member of Zn-dependent amidohydrolases that hydrolyzes nicotinamide (NAM) to nicotinic acid (NA), which is a key step in the salvage pathway of NAD(+) biosynthesis. In this paper, the catalytic mechanism of Pnc1 has been investigated by using a combined quantum-mechanical/molecular-mechanical (QM/MM) approach based on the recently obtained crystal structure of Pnc1. The reaction pathway, the detail of each elementary step, the energetics of the whole catalytic cycle, and the roles of key residues and Zn-binding site are illuminated. Our calculation results indicate that the catalytic water molecule comes from the bulk solvent, which is then deprotonated by residue D8. D8 functions as a proton transfer station between C167 and NAM, while the activated C167 serves as the nucleophile. The residue K122 only plays a role in stabilizing intermediates and transition states. The oxyanion hole formed by the amide backbone nitrogen atoms of A163 and C167 has the function to stabilize the hydroxyl anion of nicotinamide. The Zn-binding site rather than a single Zn(2+) ion acts as a Lewis acid to influence the reaction. Two elementary steps, the activation of C167 in the deamination process and the decomposition of catalytic water in the hydrolysis process, correspond to the large energy barriers of 25.7 and 28.1 kcal mol(-1), respectively, meaning that both of them contribute a lot to the overall reaction barrier. Our results may provide useful information for the design of novel and efficient Pnc1 inhibitors and related biocatalytic applications.


Assuntos
Nicotinamidase/química , Nicotinamidase/metabolismo , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Modelos Moleculares , Conformação Proteica , Teoria Quântica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Zinco/metabolismo
20.
PLoS One ; 8(12): e82705, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24340054

RESUMO

Nicotinamide mononucleotide (NMN) deamidase is one of the key enzymes of the bacterial pyridine nucleotide cycle (PNC). It catalyzes the conversion of NMN to nicotinic acid mononucleotide, which is later converted to NAD(+) by entering the Preiss-Handler pathway. However, very few biochemical data are available regarding this enzyme. This paper represents the first complete molecular characterization of a novel NMN deamidase from the halotolerant and alkaliphilic bacterium Oceanobacillus iheyensis (OiPncC). The enzyme was active over a broad pH range, with an optimum at pH 7.4, whilst maintaining 90 % activity at pH 10.0. Surprisingly, the enzyme was quite stable at such basic pH, maintaining 61 % activity after 21 days. As regard temperature, it had an optimum at 65 °C but its stability was better below 50 °C. OiPncC was a Michaelian enzyme towards its only substrate NMN, with a K m value of 0.18 mM and a kcat/K m of 2.1 mM(-1) s(-1). To further our understanding of these enzymes, a complete phylogenetic and structural analysis was carried out taking into account the two Pfam domains usually associated with them (MocF and CinA). This analysis sheds light on the evolution of NMN deamidases, and enables the classification of NMN deamidases into 12 different subgroups, pointing to a novel domain architecture never before described. Using a Logo representation, conserved blocks were determined, providing new insights on the crucial residues involved in the binding and catalysis of both CinA and MocF domains. The analysis of these conserved blocks within new protein sequences could permit the more efficient data curation of incoming NMN deamidases.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Nicotinamidase/classificação , Nicotinamidase/genética , Filogenia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Dados de Sequência Molecular , Nicotinamidase/química , Estrutura Terciária de Proteína
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