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1.
Oncol Rep ; 49(3)2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36704851

RESUMO

Colorectal carcinoma (CRC) is one of the most common types of digestive cancer. It has been reported that the ectopic expression of microRNAs (miRs) plays a critical role in the occurrence and progression of CRC. In addition, it has also been suggested that miR­151a­5p may serve as a useful biomarker for the early detection and treatment of different types of cancer and particularly CRC. However, the specific effects and underlying mechanisms of miR­151a­5p in CRC remain elusive. The results of the current study demonstrated that miR­151a­5p was upregulated in CRC cell lines and clinical tissues derived from patients with CRC. Functionally, the results showed that miR­151a­5p significantly promoted CRC cell proliferation, migration and invasion. Additionally, dual luciferase reporter assays verified that agmatinase (AGMAT) was a direct target of miR­151a­5p and it was positively associated with miR­151a­5p expression. Mechanistically, miR­151a­5p could enhance the epithelial­mesenchymal transition of CRC cells. Taken together, the results of the current study revealed a novel molecular mechanism indicating that the miR­151a­5p/AGMAT axis could serve a crucial role in the regulation of CRC and could therefore be considered as a potential therapeutic strategy for CRC.


Assuntos
Neoplasias Colorretais , MicroRNAs , Ureo-Hidrolases , Humanos , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , MicroRNAs/metabolismo , Ureo-Hidrolases/genética , Ureo-Hidrolases/metabolismo
2.
Sci Rep ; 12(1): 22088, 2022 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-36543883

RESUMO

Guanidino acids such as taurocyamine, guanidinobutyrate, guanidinopropionate, and guanidinoacetate have been detected in humans. However, except for guanidionacetate, which is a precursor of creatine, their metabolism and potential functions remain poorly understood. Agmatine has received considerable attention as a potential neurotransmitter and the human enzyme so far annotated as agmatinase (AGMAT) has been proposed as an important modulator of agmatine levels. However, conclusive evidence for the assigned enzymatic activity is lacking. Here we show that AGMAT hydrolyzed a range of linear guanidino acids but was virtually inactive with agmatine. Structural modelling and direct biochemical assays indicated that two naturally occurring variants differ in their substrate preferences. A negatively charged group in the substrate at the end opposing the guanidine moiety was essential for efficient catalysis, explaining why agmatine was not hydrolyzed. We suggest to rename AGMAT as guanidino acid hydrolase (GDAH). Additionally, we demonstrate that the GDAH substrates taurocyamine, guanidinobutyrate and guanidinopropionate were produced by human glycine amidinotransferase (GATM). The presented findings show for the first time an enzymatic activity for GDAH/AGMAT. Since agmatine has frequently been proposed as an endogenous neurotransmitter, the current findings clarify important aspects of the metabolism of agmatine and guanidino acid derivatives in humans.


Assuntos
Guanidinas , Ureo-Hidrolases , Humanos , Agmatina/metabolismo , Guanidinas/metabolismo , Hidrólise , Ureo-Hidrolases/metabolismo
3.
Plant Cell Physiol ; 63(9): 1298-1308, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-35861030

RESUMO

Seed dormancy is a very complex trait controlled by interactions between genetic and environmental factors. Nitrate is inversely correlated with seed dormancy in Arabidopsis. This is explained by the fact that seed dry storage (after-ripening) reduces the need for nitrogen for germination. When nitrate is absorbed by plants, it is first reduced to nitrite and then to ammonium for incorporation into amino acids, nucleic acids and chlorophyll. Previously, we showed that ALLANTOATE AMIDOHYDROLASE (AtAAH) transcripts are up-regulated in imbibed dormant seeds compared with after-ripened seeds. AAH is an enzyme in the uric acid catabolic pathway which catalyzes the hydrolysis of allantoate to yield CO2, NH3 and S-ureidoglycine. This pathway is the final stage of purine catabolism, and functions in plants and some bacteria to provide nitrogen, particularly when other nitrogen sources are depleted. Ataah mutant seeds are more dormant and accumulate high levels of allantoate, allantoin and urea, whereas energy-related metabolites and several amino acids are lower upon seed imbibition in comparison with Columbia-0. AtAAH expression could be detected during the early stages of seed development, with a transient increase around 8 d after pollination. AtAAH expression is the highest in mature pollen. The application of exogenous potassium nitrate can partly complement the higher dormancy phenotype of the Ataah mutant seeds, whereas other nitrogen sources cannot. Our results indicate that potassium nitrate does not specifically overcome the alleviated dormancy levels in Ataah mutant seeds, but promotes germination in general. Possible pathways by which AtAAH affects seed germination are discussed.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Aminoácidos/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação , Nitratos/metabolismo , Nitratos/farmacologia , Nitrogênio/metabolismo , Dormência de Plantas/genética , Compostos de Potássio , Sementes/metabolismo , Ureo-Hidrolases
4.
Sheng Wu Gong Cheng Xue Bao ; 38(12): 4601-4614, 2022 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-36593196

RESUMO

Creatinine levels in biological fluids are important indicators for the clinical evaluation of renal function. Creatinase (CRE, EC3.5.3.3) is one of the key enzymes in the enzymatic measurement of creatinine concentration, and it is also the rate-limiting enzyme in the whole enzymatic cascade system. The poor catalytic activity of CRE severely limits its clinical and industrial applications. To address this issue, a semi-rational design is applied to increase the activity of a creatinase from Alcaligenes sp. KS-85 (Al-CRE). By high-throughput screen of saturation mutagenesis libraries on the selected hotspot mutations, multiple variant enzymes with increased activity are obtained. The five-point best variant enzyme (I304L/F395V/K351V/Y63S/Q88A) were further obtained by recombine the improved mutations sites that to showed a 2.18-fold increased specific activity. Additionally, structure analysis is conducted to understand the mechanism of the activity change. This study paves the way for a better practical application of creatinase and may help further understand its catalytic mechanism.


Assuntos
Ureo-Hidrolases , Creatinina , Mutagênese Sítio-Dirigida , Ureo-Hidrolases/genética , Catálise
5.
Chem Commun (Camb) ; 57(38): 4726-4729, 2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-33977964

RESUMO

A nano-integrated portable enzymatic microfluidic electrochemical biochip was developed for single-step point-of-care testing of creatinine. The biochip could automatically eliminate a lot of interferences from practical biological samples and enzymatic intermediate products. Gold nanostructure- and carbon nanotube-based screen-printed carbon electrodes were integrated into microfluidic structures to improve the detection performance for creatinine. The microfluidic electrochemical biochip holds promise to become a practical device for medical diagnosis, especially POCT.


Assuntos
Creatinina/sangue , Técnicas Eletroquímicas , Dispositivos Lab-On-A-Chip , Nanotecnologia , Sistemas Automatizados de Assistência Junto ao Leito , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Tamanho da Partícula , Sarcosina Oxidase/metabolismo , Ureo-Hidrolases/metabolismo
6.
Int J Mol Sci ; 22(9)2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946272

RESUMO

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer's, Parkinson's, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Ureo-Hidrolases/química , Agmatina/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Especificidade por Substrato , Ureo-Hidrolases/metabolismo
7.
PLoS One ; 16(4): e0248991, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33857156

RESUMO

Agmatine amidinohydrolase, or agmatinase, catalyzes the conversion of agmatine to putrescine and urea. This enzyme is found broadly across kingdoms of life and plays a critical role in polyamine biosynthesis and the regulation of agmatine concentrations. Here we describe the high-resolution X-ray crystal structure of the E. coli agmatinase, SPEB. The data showed a relatively high degree of pseudomerohedral twinning, was ultimately indexed in the P31 space group and led to a final model with eighteen chains, corresponding to three full hexamers in the asymmetric unit. There was a solvent content of 38.5% and refined R/Rfree values of 0.166/0.216. The protein has the conserved fold characteristic of the agmatine ureohydrolase family and displayed a high degree of structural similarity among individual protomers. Two distinct peaks of electron density were observed in the active site of most of the eighteen chains of SPEB. As the activity of this protein is known to be dependent upon manganese and the fold is similar to other dinuclear metallohydrolases, these peaks were modeled as manganese ions. The orientation of the conserved active site residues, in particular those amino acids that participate in binding the metal ions and a pair of acidic residues (D153 and E274 in SPEB) that play a role in catalysis, are similar to other agmatinase and arginase enzymes and is consistent with a hydrolytic mechanism that proceeds via a metal-activated hydroxide ion.


Assuntos
Proteínas de Escherichia coli/química , Ureo-Hidrolases/química , Domínio Catalítico , Sequência Conservada , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Ureo-Hidrolases/metabolismo
8.
Front Immunol ; 12: 626840, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33717145

RESUMO

Despite significant advances in prevention and treatment of transplant rejection with immunosuppressive medications, we continue to face challenges of long-term graft survival, detrimental medication side effects to both the recipient and transplanted organ together with risks for opportunistic infections. Transplantation tolerance has so far only been achieved through hematopoietic chimerism, which carries with it a serious and life-threatening risk of graft versus host disease, along with variability in persistence of chimerism and uncertainty of sustained tolerance. More recently, numerous in vitro and in vivo studies have explored the therapeutic potential of silent clearance of apoptotic cells which have been well known to aid in maintaining peripheral tolerance to self. Apoptotic cells from a donor not only have the ability of down regulating the immune response, but also are a way of providing donor antigens to recipient antigen-presenting-cells that can then promote donor-specific peripheral tolerance. Herein, we review both laboratory and clinical evidence that support the utility of apoptotic cell-based therapies in prevention and treatment of graft versus host disease and transplant rejection along with induction of donor-specific tolerance in solid organ transplantation. We have highlighted the potential limitations and challenges of this apoptotic donor cell-based therapy together with ongoing advancements and attempts made to overcome them.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Rejeição de Enxerto/imunologia , Doença Enxerto-Hospedeiro/imunologia , Imunossupressores/uso terapêutico , Transplante de Órgãos , Animais , Células Apresentadoras de Antígenos/transplante , Apoptose , Quimerismo , Rejeição de Enxerto/prevenção & controle , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Doadores de Tecidos , Imunologia de Transplantes , Tolerância ao Transplante , Ureo-Hidrolases/imunologia
9.
Fungal Genet Biol ; 146: 103496, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33290821

RESUMO

During the infection and colonization process, the rice blast fungus Magnaporthe oryzae faces various challenges from hostile environment, such as nutrient limitation and carbon stress, while carbon catabolite repression (CCR) mechanism would facilitate the fungus to shrewdly and efficiently utilize carbon nutrients under fickle nutritional conditions since it ensures the preferential utilization of most preferred carbon sources through repressing the expression of enzymes required for the utilization of less preferred carbon sources. Researches on M. oryzae CCR have made some progress, however the involved regulation mechanism is still largely obscured, especially, little is known about the key carbon catabolite repressor CreA. Here we identified and characterized the biological functions of the CreA homolog MoCreA in M. oryzae. MoCreA is constitutively expressed throughout all the life stages of the fungus, and it can shuttle between nucleus and cytoplasm which is induced by glucose. Following functional analyses of MoCreA suggested that it was required for the vegetative growth, conidiation, appressorium formation and pathogenicity of M. oryzae. Moreover, comparative transcriptomic analysis revealed that disruption of MoCreA resulted in the extensive gene expression variations, including a large number of carbon metabolism enzymes, transcription factors and pathogenicity-related genes. Taken together, our results demonstrated that, as a key regulator of CCR, MoCreA plays a vital role in precise regulation of the asexual development and pathogenicity of the rice blast fungus.


Assuntos
Ascomicetos/genética , Repressão Catabólica/genética , Reprodução Assexuada/genética , Fatores de Transcrição/genética , Ascomicetos/patogenicidade , Carbono/metabolismo , Citoplasma/genética , Proteínas Fúngicas , Glucose/metabolismo , Oryza/genética , Oryza/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Esporos Fúngicos/genética , Esporos Fúngicos/patogenicidade , Ureo-Hidrolases/genética , Virulência/genética
10.
Neurosci Lett ; 740: 135447, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33127446

RESUMO

Alzheimer's disease is an age related progressive neurodegenerative disorder characterized by decline in cognitive functions, such as memory loss and behavioural abnormalities. The present study sought to assess alterations in agmatine metabolism in the beta-amyloid (Aß1-42) Alzheimer's disease mouse model. Aß1-42 injected mice showed impairment of cognitive functioning as evidenced by increased working and reference memory errors in radial arm maze (RAM). This cognitive impairment was associated with a reduction in the agmatine levels and elevation in its degrading enzyme, agmatinase, whereas reduced immunocontent was observed in its synthesizing enzyme arginine decarboxylase expression within hippocampus and prefrontal cortex. Chronic agmatine treatment and its endogenous modulation by l-arginine, or arcaine or aminoguanidine prevented the learning and memory impairment induced by single intracranial Aß1-42 peptide injection. In conclusion, the present study suggests the importance of the endogenous agmatinergic system in ß-amyloid induced memory impairment in mice.


Assuntos
Agmatina/metabolismo , Agmatina/farmacologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides , Transtornos da Memória/metabolismo , Fragmentos de Peptídeos , Doença de Alzheimer/induzido quimicamente , Doença de Alzheimer/psicologia , Animais , Carboxiliases/biossíntese , Transtornos Cognitivos/induzido quimicamente , Transtornos Cognitivos/psicologia , Hipocampo/enzimologia , Masculino , Aprendizagem em Labirinto , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/psicologia , Camundongos , Córtex Pré-Frontal/enzimologia , Desempenho Psicomotor/efeitos dos fármacos , Ureo-Hidrolases/metabolismo
11.
Fungal Genet Biol ; 157: 103634, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34634482

RESUMO

Agmatinase is a metallohydrolase involved in the hydrolysis of agmatine to produce urea and putrescine. Although its role in organisms is still under study, there are no reports of this family of enzymes in filamentous fungi. Recently, a protein showing agmatinase activity was reported in Neurospora crassa. Therefore, the aim of this work is to determine if the protein (AGM-1) found in the filamentous fungus N. crassa is a true agmatinase. The protein AGM-1was purified directly from N. crassa cultures, and its enzymatic characterization was carried out. The catalytic parameters such as optimum pH, thermostability, transformation kinetics, and activity in the presence of a cofactor were determined. The results show that AGM-1 can use manganese as a cofactor for its enzymatic activity, showing a transformation rate constant (kcat) of 77 s-1 and an affinity constant (KM) of 50.5 mM. The protein loses 50% of its activity when incubated 15 min at 30 °C and reaches maximal enzymatic activity at a pH range of 8-8.5. Our results indicate that the AGM-1 from N. crassa shows similar characteristics to true agmatinases already reported in other organisms. Thus, our findings strongly support that the protein annotated as hypothetical agmatinase in N. crassa is a true agmatinase.


Assuntos
Agmatina , Neurospora crassa , Catálise , Neurospora crassa/genética , Ureo-Hidrolases
12.
Biosensors (Basel) ; 11(9)2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34562932

RESUMO

Creatinine has become an important indicator for the early detection of uremia. However, due to the disadvantages of external power supply and large volume, some commercial devices for detecting creatinine concentration have lost a lot of popularity in everyday life. This paper describes the development of a self-powered biosensor for detecting creatinine in sweat. The biosensor can detect human creatinine levels in real time without the need for an external power source, providing information about the body's overall health. The piezoelectric output voltage of creatininase/creatinase/sarcosine oxidase-modified ZnO nanowires (NWs) is significantly dependent on the creatinine concentration due to the coupling effect of the piezoelectric effect and enzymatic reaction (piezo-enzymatic-reaction effect), which can be regarded as both electrical energy and biosensing signal. Our results can be used for the detection of creatinine levels in the human body and have great potential in the prediction of related diseases.


Assuntos
Técnicas Biossensoriais , Nanofios , Amidoidrolases , Creatinina , Fontes de Energia Elétrica , Eletricidade , Humanos , Suor , Ureo-Hidrolases , Óxido de Zinco/química
13.
Microb Cell Fact ; 19(1): 188, 2020 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-33008411

RESUMO

L-rhamnose (6-deoxy-mannose) occurs in nature mainly as a component of certain plant structural polysaccharides and bioactive metabolites but has also been found in some microorganisms and animals. The release of L-rhamnose from these substrates is catalysed by extracellular enzymes including α-L-rhamnosidases, the production of which is induced in its presence. The free sugar enters cells via specific uptake systems where it can be metabolized. Of two L-rhamnose catabolic pathways currently known in microorganisms a non-phosphorylated pathway has been identified in fungi and some bacteria but little is known of the regulatory mechanisms governing it in fungi. In this study two genes (lraA and lraB) are predicted to be involved in the catabolism of L-rhamnose, along with lraC, in the filamentous fungus Aspergillus nidulans. Transcription of all three is co-regulated with that of the genes encoding α-L-rhamnosidases, i.e. induction mediated by the L-rhamnose-responsive transcription factor RhaR and repression of induction in the presence of glucose via a CreA-independent mechanism. The participation of lraA/AN4186 (encoding L-rhamnose dehydrogenase) in L-rhamnose catabolism was revealed by the phenotypes of knock-out mutants and their complemented strains. lraA deletion negatively affects both growth on L-rhamnose and the synthesis of α-L-rhamnosidases, indicating not only the indispensability of this pathway for L-rhamnose utilization but also that a metabolite derived from this sugar is the true physiological inducer.


Assuntos
Aspergillus nidulans/metabolismo , Proteínas Fúngicas/genética , Glucose/metabolismo , Ramnose/metabolismo , Ureo-Hidrolases/metabolismo , Aspergillus nidulans/genética , Desidrogenases de Carboidrato/genética , Desidrogenases de Carboidrato/metabolismo , Regulação Fúngica da Expressão Gênica , Redes e Vias Metabólicas , Fosforilação , Fatores de Transcrição
14.
Microb Cell Fact ; 19(1): 194, 2020 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-33069232

RESUMO

BACKGROUND: Enzymatic quantification of creatinine has become an essential method for clinical evaluation of renal function. Although creatinase (CR) is frequently used for this purpose, its poor thermostability severely limits industrial applications. Herein, we report a novel creatinase from Alcaligenes faecalis (afCR) with higher catalytic activity and lower KM value, than currently used creatinases. Furthermore, we developed a non-biased phylogenetic consensus method to improve the thermostability of afCR. RESULTS: We applied a non-biased phylogenetic consensus method to identify 59 candidate consensus residues from 24 creatinase family homologs for screening afCR mutants with improved thermostability. Twenty-one amino acids of afCR were selected to mutagenesis and 11 of them exhibited improved thermostability compared to the parent enzyme (afCR-M0). Combination of single-site mutations in sequential screens resulted in a quadruple mutant D17V/T199S/L6P/T251C (M4-2) which showed ~ 1700-fold enhanced half-life at 57 °C and a 4.2 °C higher T5015 than that of afCR-M0. The mutant retained catalytic activity equivalent to afCR-M0, and thus showed strong promise for application in creatinine detection. Structural homology modeling revealed a wide range of potential molecular interactions associated with individual mutations that contributed to improving afCR thermostability. CONCLUSIONS: Results of this study clearly demonstrated that the non-biased-phylogenetic consensus design for improvement of thermostability in afCR is effective and promising in improving the thermostability of more enzymes.


Assuntos
Alcaligenes faecalis/enzimologia , Mutagênese Sítio-Dirigida/métodos , Temperatura , Ureo-Hidrolases/metabolismo , Substituição de Aminoácidos , Estabilidade Enzimática , Cinética , Simulação de Dinâmica Molecular , Filogenia , Engenharia de Proteínas , Ureo-Hidrolases/genética
15.
mBio ; 11(4)2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32788384

RESUMO

Amino acid metabolism is crucial for fungal growth and development. Ureohydrolases produce amines when acting on l-arginine, agmatine, and guanidinobutyrate (GB), and these enzymes generate ornithine (by arginase), putrescine (by agmatinase), or GABA (by 4-guanidinobutyrase or GBase). Candida albicans can metabolize and grow on arginine, agmatine, or guanidinobutyrate as the sole nitrogen source. Three related C. albicans genes whose sequences suggested that they were putative arginase or arginase-like genes were examined for their role in these metabolic pathways. Of these, Car1 encoded the only bona fide arginase, whereas we provide evidence that the other two open reading frames, orf19.5862 and orf19.3418, encode agmatinase and guanidinobutyrase (Gbase), respectively. Analysis of strains with single and multiple mutations suggested the presence of arginase-dependent and arginase-independent routes for polyamine production. CAR1 played a role in hyphal morphogenesis in response to arginine, and the virulence of a triple mutant was reduced in both Galleria mellonella and Mus musculus infection models. In the bloodstream, arginine is an essential amino acid that is required by phagocytes to synthesize nitric oxide (NO). However, none of the single or multiple mutants affected host NO production, suggesting that they did not influence the oxidative burst of phagocytes.IMPORTANCE We show that the C. albicans ureohydrolases arginase (Car1), agmatinase (Agt1), and guanidinobutyrase (Gbu1) can orchestrate an arginase-independent route for polyamine production and that this is important for C. albicans growth and survival in microenvironments of the mammalian host.


Assuntos
Agmatina/metabolismo , Arginina/metabolismo , Candida albicans/enzimologia , Candida albicans/patogenicidade , Proteínas Fúngicas/metabolismo , Ureo-Hidrolases/metabolismo , Aminoácidos/metabolismo , Animais , Arginase/genética , Arginase/metabolismo , Clonagem Molecular , Feminino , Larva/microbiologia , Redes e Vias Metabólicas , Camundongos , Camundongos Endogâmicos BALB C , Mariposas/microbiologia , Células RAW 264.7 , Ureo-Hidrolases/genética , Virulência
16.
Microb Cell Fact ; 19(1): 155, 2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-32727458

RESUMO

BACKGROUND: Filamentous fungi have long been used as hosts for the production of proteins, enzymes and valuable products in various biotechnological applications. However, recombinant proteins are expressed with highly secreted host proteins when stronger promoters are used under inducing conditions. In addition, the efficiency of target protein expression can be limited by the application of constitutive promoters in recently developed filamentous fungal expression systems. RESULTS: In this study, a novel expression system was constructed by using a Penicillium oxalium strain that has powerful protein secretion capability. The secretory background of the host was reduced by knocking out the Amy13A protein and utilizing the starch as a carbon source. The strong promoter amy15A(p) was further improved by overexpressing the transcription activator AmyR and deleting of putative repressor CreA. By using the native amylase Amy15A as a reporter, the efficiency of expression from the amy15A promoter was dramatically and specifically enhanced after redesigning the regulatory network of amylase expression. CONCLUSIONS: Our researches clearly indicated that the triple-gene recombinant strain Δ13A-OamyR-ΔCreA, with the amy15A(p) promoter could be used as a suitable expression system especially for high-level and high-purity protein production.


Assuntos
Amilases/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Penicillium/genética , Amilases/biossíntese , Penicillium/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Amido/metabolismo , Fatores de Transcrição/genética , Ureo-Hidrolases/genética
17.
Int J Mycobacteriol ; 9(2): 138-143, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32474534

RESUMO

Background: Polyamines are widespread intracellular molecules able to influence antibiotic susceptibility, but almost nothing is known on their occurrence and physiological role in mycobacteria. Methods: here, we analyzed transcriptomic, proteomic and biochemical data and obtained the first evidence for the post-transcriptional expression of some genes attributed to polyamine metabolism and polyamine transport in Mycolicibacterium smegmatis (basionym Mycobacterium smegmatis). Results: in our experiments, exponentially growing cells demonstrated transcription of 21 polyamine-associated genes and possessed 7 enzymes of polyamine metabolism and 2 polyamine transport proteins. Conclusion: Mycolicibacterium smegmatis putrescine synthesizing enzyme agmatinase SpeB was originally shown to catalyze agmatine conversion to putrescine in vitro. Nevertheless, we have not found any polyamines in mycobacterial cells.


Assuntos
Mycobacterium smegmatis/química , Mycobacterium smegmatis/enzimologia , Poliaminas/análise , Ureo-Hidrolases/metabolismo , Agmatina/metabolismo , Perfilação da Expressão Gênica , Mycobacterium smegmatis/genética , Proteômica , Putrescina/metabolismo , Ureo-Hidrolases/genética
18.
Int J Mol Sci ; 21(11)2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-32531922

RESUMO

Agmatine is a neurotransmitter with anticonvulsant, anti-neurotoxic and antidepressant-like effects, in addition it has hypoglycemic actions. Agmatine is converted to putrescine and urea by agmatinase (AGM) and by an agmatinase-like protein (ALP), a new type of enzyme which is present in human and rodent brain tissues. Recombinant rat brain ALP is the only mammalian protein that exhibits significant agmatinase activity in vitro and generates putrescine under in vivo conditions. ALP, despite differing in amino acid sequence from all members of the ureohydrolase family, is strictly dependent on Mn2+ for catalytic activity. However, the Mn2+ ligands have not yet been identified due to the lack of structural information coupled with the low sequence identity that ALPs display with known ureohydrolases. In this work, we generated a structural model of the Mn2+ binding site of the ALP and we propose new putative Mn2+ ligands. Then, we cloned and expressed a sequence of 210 amino acids, here called the "central-ALP", which include the putative ligands of Mn2+. The results suggest that the central-ALP is catalytically active, as agmatinase, with an unaltered Km for agmatine and a decreased kcat. Similar to wild-type ALP, central-ALP is activated by Mn2+ with a similar affinity. Besides, a simple mutant D217A, a double mutant E288A/K290A, and a triple mutant N213A/Q215A/D217A of these putative Mn2+ ligands result on the loss of ALP agmatinase activity. Our results indicate that the central-ALP contains the active site for agmatine hydrolysis, as well as that the residues identified are relevant for the ALP catalysis.


Assuntos
Agmatina/metabolismo , Manganês/metabolismo , Ureo-Hidrolases/química , Ureo-Hidrolases/metabolismo , Animais , Sítios de Ligação , Escherichia coli/genética , Cinética , Mamíferos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , Temperatura , Ureo-Hidrolases/genética
19.
Anal Biochem ; 595: 113618, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32057730

RESUMO

Ureohydrolases are members of the metallohydrolase family of enzymes. Here, a simple continuous assay for agmatinase (AGM) activity was established by following the degradation of agmatine to urea and putrescine using isothermal titration calorimetry (ITC). ITC is particularly useful for kinetic assays when substrates of interest do not possess suitable chromophores that facilitate the continuous spectrophotometric detection of substrate depletion and/or product formation. In order to assess the accuracy of the ITC-based assay, catalytic parameters were also determined using a discontinuous, colorimetric assay. Both methods resulted in comparable kinetic parameters. From the colorimetric assay the kcat and KM values are 131 s-1 and 0.25 mM, respectively, and from the ITC assay the corresponding parameters are 30 s-1 and 0.45 mM, respectively. The continuous ITC-based assay will facilitate functional studies for an enzyme that is an emerging target for the development of addiction treatments.


Assuntos
Biocatálise , Calorimetria , Ureo-Hidrolases/metabolismo , Escherichia coli/enzimologia , Hidrólise , Cinética , Modelos Moleculares , Ureo-Hidrolases/química , Ureo-Hidrolases/isolamento & purificação
20.
Environ Microbiol ; 22(1): 107-121, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31608522

RESUMO

Cellulose is a by-product of agricultural production and an abundant waste. As a carbon source, cellulose can be degraded and utilized by fungi. Carbon sources, which act as nutrients, not only provide energy but also serve as regulators of gene expression, metabolism and growth, through various signalling networks that enable cells to sense and adapt to varying environmental conditions. Nutrient-sensing pathways prioritize the use of preferred carbon sources and regulate the production of cellulose-degrading enzymes when necessary. Understanding the regulation of the fungal cellulolytic response will become increasingly important because we strive to increase the efficiency of the utilization of these renewable energy sources. Here, we show that Glsnf1, a sucrose-nonfermenting serine-threonine-protein kinase 1 (Snf1)/AMP-activated protein kinase homologue in medicinal macro basidiomycete Ganoderma lucidum, actively responds to carbon alterations and positively regulates cellulase activity and cellulase-related gene transcription. The carbon catabolite repressor CreA, a zinc binuclear cluster transcription factor that mediates the sensing of nutrients and suppression of the transcription of a number of genes necessary for the consumption of a less preferred carbon source, participates in the Glsnf1-mediated regulation of cellulases. Glsnf1 not only negatively regulates the transcription level of the CreA gene but also hinders its localization in the nucleus. Overall, our findings reveal a key nutrient-sensing mechanism that is critical for the modulation of carbon source adaptation in G. lucidum.


Assuntos
Celulose/metabolismo , Proteínas Fúngicas , Proteínas Serina-Treonina Quinases/metabolismo , Reishi/genética , Reishi/metabolismo , Ureo-Hidrolases , Metabolismo dos Carboidratos/genética , Carbono/metabolismo , Celulase/genética , Celulase/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Ureo-Hidrolases/genética , Ureo-Hidrolases/metabolismo
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