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1.
Parasit Vectors ; 13(1): 200, 2020 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-32306993

RESUMO

BACKGROUND: Approximately 30% of children worldwide are infected with gastrointestinal parasites. Depending on the species, parasites can disrupt intestinal bacterial microbiota affecting essential vitamin biosynthesis. METHODS: Stool samples were collected from 37 asymptomatic children from a previous cross-sectional Argentinian study. A multi-parallel real-time quantitative PCR was implemented for Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Trichuris trichiura, Cryptosporidium spp., Entamoeba histolytica and Giardia duodenalis. In addition, whole-genome sequencing analysis was conducted for bacterial microbiota on all samples and analyzed using Livermore Metagenomic Analysis Toolkit and DIAMOND software. Separate analyses were carried out for uninfected, Giardia-only, Giardia + helminth co-infections, and helminth-only groups. RESULTS: For Giardia-only infected children compared to uninfected children, DNA sequencing data showed a decrease in microbiota biodiversity that correlated with increasing Giardia burden and was statistically significant using Shannon's alpha diversity (Giardia-only > 1 fg/µl 2.346; non-infected group 3.253, P = 0.0317). An increase in diversity was observed for helminth-only infections with a decrease in diversity for Giardia + helminth co-infections (P = 0.00178). In Giardia-only infections, microbiome taxonomy changed from Firmicutes towards increasing proportions of Prevotella, with the degree of change related to the intensity of infection compared to uninfected (P = 0.0317). The abundance of Prevotella bacteria was decreased in the helminths-only group but increased for Giardia + helminth co-infections (P = 0.0262). Metagenomic analysis determined cobalamin synthesis was decreased in the Giardia > 1 fg/µl group compared to both the Giardia < 1 fg/µl and the uninfected group (P = 0.0369). Giardia + helminth group also had a decrease in cobalamin CbiM genes from helminth-only infections (P = 0.000754). CONCLUSION: The study results may provide evidence for an effect of parasitic infections enabling the permissive growth of anaerobic bacteria such as Prevotella, suggesting an altered capacity of vitamin B12 (cobalamin) biosynthesis and potential impact on growth and development in children .


Assuntos
Coinfecção , Microbioma Gastrointestinal/genética , Intestinos , Parasitos/genética , Vitamina B 12/genética , Animais , Criança , Pré-Escolar , Coinfecção/microbiologia , Coinfecção/parasitologia , Estudos Transversais , DNA de Helmintos , DNA de Protozoário , Feminino , Genes Bacterianos , Giardia lamblia/classificação , Giardia lamblia/genética , Giardia lamblia/isolamento & purificação , Helmintos/classificação , Helmintos/genética , Helmintos/isolamento & purificação , Humanos , Intestinos/microbiologia , Intestinos/parasitologia , Masculino , Metagenômica , Parasitos/classificação , Parasitos/isolamento & purificação , Projetos Piloto , Reação em Cadeia da Polimerase em Tempo Real , Vitamina B 12/metabolismo , Sequenciamento Completo do Genoma
2.
PLoS One ; 15(1): e0224646, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31905202

RESUMO

Twelve human THAP proteins share the THAP domain, an evolutionary conserved zinc-finger DNA-binding domain. Studies of different THAP proteins have indicated roles in gene transcription, cell proliferation and development. We have analyzed this protein family, focusing on THAP7 and THAP11. We show that human THAP proteins possess differing homo- and heterodimer formation properties and interaction abilities with the transcriptional co-regulator HCF-1. HEK-293 cells lacking THAP7 were viable but proliferated more slowly. In contrast, HEK-293 cells were very sensitive to THAP11 alteration. Nevertheless, HEK-293 cells bearing a THAP11 mutation identified in a patient suffering from cobalamin disorder (THAP11F80L) were viable although proliferated more slowly. Cobalamin disorder is an inborn vitamin deficiency characterized by neurodevelopmental abnormalities, most often owing to biallelic mutations in the MMACHC gene, whose gene product MMACHC is a key enzyme in the cobalamin (vitamin B12) metabolic pathway. We show that THAP11F80L selectively affected promoter binding by THAP11, having more deleterious effects on a subset of THAP11 targets, and resulting in altered patterns of gene expression. In particular, THAP11F80L exhibited a strong effect on association with the MMACHC promoter and led to a decrease in MMACHC gene transcription, suggesting that the THAP11F80L mutation is directly responsible for the observed cobalamin disorder.


Assuntos
Oxirredutases/genética , Proteínas Repressoras/genética , Deficiência de Vitamina B 12/genética , Vitamina B 12/genética , Linhagem Celular , Proliferação de Células/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Fator C1 de Célula Hospedeira/genética , Humanos , Redes e Vias Metabólicas/genética , Mutação/genética , Regiões Promotoras Genéticas , Ligação Proteica/genética , Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/patologia
3.
Food Chem ; 312: 126064, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31891887

RESUMO

Biophysical insight into the binding interaction between the major whey protein, ß-Lactoglobulin (ßLG) and vitamin B12, was studied using different spectroscopic tools such as steady-state & time-resolved fluorescence spectroscopy, Circular Dichroism (CD) and Fluorescence Correlation Spectroscopy (FCS). The intrinsic fluorescence of ßLG was quenched by vitamin B12. From the time-resolved fluorescence experiment, the nature of quenching was found to be static suggesting ground-state complex formation between ßLG and vitamin B12, which was also supported by the excitation spectra. Synchronous fluorescence spectra revealed that the tryptophan residue microenvironment of ßLG was affected by the vitamin B12. The CD spectra suggested that the secondary structure of the ßLG remains unaffected by vitamin B12. From the FCS experiment, the tertiary structure of ßLG was observed to be stable in the presence of vitamin B12 at the single-molecule level. The outcome of this study might have potential applications in the food and pharmaceutical industry.


Assuntos
Lactoglobulinas/química , Vitamina B 12/química , Fenômenos Biofísicos , Dicroísmo Circular , Lactoglobulinas/metabolismo , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Triptofano/química , Vitamina B 12/metabolismo
4.
BMC Med Genet ; 21(1): 12, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31931749

RESUMO

BACKGROUND: Gaucher disease (GD) is a lysosomal disorder caused by biallelic pathogenic mutations in the GBA1 gene that encodes beta-glucosidase (GCase), and more rarely, by a deficiency in the GCase activator, saposin C. Clinically, GD manifests with heterogeneous multiorgan involvement mainly affecting hematological, hepatic and neurological axes. This disorder is divided into three types, based on the absence (type I) or presence and severity (types II and III) of involvement of the central nervous system. At the cellular level, deficiency of GBA1 disturbs lysosomal storage with buildup of glucocerebroside. The consequences of disturbed lysosomal metabolism on biochemical pathways that require lysosomal processing are unknown. Abnormal systemic markers of cobalamin (Cbl, B12) metabolism have been reported in patients with GD, suggesting impairments in lysosomal handling of Cbl or in its downstream utilization events. METHODS: Cultured skin fibroblasts from control humans (n = 3), from patients with GD types I (n = 1), II (n = 1) and III (n = 1) and an asymptomatic carrier of GD were examined for their GCase enzymatic activity and lysosomal compartment intactness. Control human and GD fibroblasts were cultured in growth medium with and without 500 nM hydroxocobalamin supplementation. Cellular cobalamin status was examined via determination of metabolomic markers in cell lysate (intracellular) and conditioned culture medium (extracellular). The presence of transcobalamin (TC) in whole cell lysates was examined by Western blot. RESULTS: Cultured skin fibroblasts from GD patients exhibited reduced GCase activity compared to healthy individuals and an asymptomatic carrier of GD, demonstrating a preserved disease phenotype in this cell type. The concentrations of total homocysteine (tHcy), methylmalonic acid (MMA), cysteine (Cys) and methionine (Met) in GD cells were comparable to control levels, except in one patient with GD III. The response of these metabolomic markers to supplementation with hydroxocobalamin (HOCbl) yielded variable results. The content of transcobalamin in whole cell lysates was comparable in control human and GD patients. CONCLUSIONS: Our results indicate that cobalamin transport and cellular processing pathways are overall protected from lysosomal storage damage in GD fibroblasts. Extending these studies to hepatocytes, macrophages and plasma will shed light on cell- and compartment-specific vitamin B12 metabolism in Gaucher disease.


Assuntos
Doença de Gaucher/genética , Glucosilceramidase/genética , Vitamina B 12/metabolismo , beta-Glucosidase/genética , Técnicas de Cultura de Células , Feminino , Fibroblastos/metabolismo , Doença de Gaucher/metabolismo , Doença de Gaucher/patologia , Homocisteína/metabolismo , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Masculino , Ácido Metilmalônico/metabolismo , Mutação , Fenótipo , Saposinas/genética , Transcobalaminas/metabolismo
5.
Environ Pollut ; 259: 113947, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31931416

RESUMO

In this study, the YH consortium, an ethene-producing culture, was used to evaluate the effect of vitamin B12 (VB12) on trichloroethene (TCE) dechlorination by transferring the original TCE-reducing culture with or without adding exogenous VB12. Ultra-high performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) was applied to detect the concentrations of VB12 and its lower ligand 5,6-dimethylbenzimidazole (DMB) in the cultures. After three successive VB12 starvation cycles, the dechlorination of TCE stopped mostly at cis-dichloroethene (cDCE), and no ethene was found; methane production increased significantly, and no VB12 was detected. Results suggest that the co-cultured microbes may not be able to provide enough VB12 as a cofactor for the growth of Dehalococcoides in the YH culture, possibly due to the competition for corrinoids between Dehalococcoides and methanogens. The relative abundances of 16 S rRNA gene of Dehalococcoides and reductive dehalogenase genes tceA or vcrA were lower in the cultures without VB12 compared with the cultures with VB12. VB12 limitation changed the microbial community structures of the consortia. In the absence of VB12, the microbial community shifted from dominance of Chloroflexi to Proteobacteria after three consecutive VB12 starvation cycles, and the dechlorinating genus Dehalococcoides declined from 42.9% to 13.5%. In addition, Geobacter, Clostridium, and Desulfovibrio were also present in the cultures without VB12. Furthermore, the abundance of archaea increased under VB12 limited conditions. Methanobacterium and Methanosarcina were the predominant archaea in the culture without VB12.


Assuntos
Biodegradação Ambiental , Chloroflexi , Tricloroetileno/metabolismo , Vitamina B 12/metabolismo , Anaerobiose , Cromatografia Líquida , Espectrometria de Massas em Tandem , Vitaminas
6.
Biochimie ; 168: 185-189, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31715214

RESUMO

Lactobacillus coryniformis CRL 1001 and L. reuteri CRL 1098 have the complete genes necessary to synthesize pseudo-cobalamin as final product in a vitamin B12 free commercial medium. Unlike vitaminB12 (the most biologically active form), the pseudo-cobalamin contains adenine instead of 5,6-dimethlbenzimidazole (DMB) in the Coα-ligand. Considering the vitamin B12-gene clusters of these bacteria, the aim of this work was to analyze the production of corrinoids with DMB (vitamin B12) instead of adenine (pseudo-B12) as lower ligand base in a vitamin B12 free chemically defined medium (CDM) without purines. Genome-wide screening of genes related to purine metabolism showed that both strains possess all pur genes necessary for the synthesis of inositol monophosphate, the main precursor for purine biosynthesis. Accordingly, both strains were able to grow in B12 free CDM without purines, with the supplementation of different synthetic intermediaries. Isolated compounds with positive vitamin B12 activity were quantified and characterized by LC/MS-MS. Total corrinoids values were higher for both strains in comparison to those obtained in vitaminB12 free commercial medium. Interestingly, CRL 1001 strain synthesized cobalamin, suggesting that this strain is able to activate DMB as nitrogenous base instead adenine when it is in excess in a purine-free medium. The present paper represents the first demonstration of a partial metabolic shift to produce vitamin B12 in a Lactobacillus strain.


Assuntos
Lactobacillus reuteri/metabolismo , Lactobacillus/metabolismo , Purinas/metabolismo , Vitamina B 12/análogos & derivados , Vitamina B 12/metabolismo , Meios de Cultura/metabolismo , Redes e Vias Metabólicas
7.
Int J Vitam Nutr Res ; 90(1-2): 131-140, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30758268

RESUMO

Background: Some micronutrients like folate, vitamin B12, B6, and B2 are the source of coenzymes, which participate in one-carbon metabolism. Any disruption in this metabolism can interfere with DNA replication, repair and regulation of gene expression and ultimately promote the likelihood of carcinogenesis. This study aimed at investigating the relationship between the intakes of micronutrients involved in one-carbon metabolism with breast cancer (BrCa) and its subtype's odds. Methods: Nutrients' intake from diet and supplements were collected through interviewing 151 cases and 154 controls by a 168-item semiquantitative food frequency questionnaire. Logistic regression was used to determine the relationship between dietary and/or total intake of studied nutrients and odds of BrCa and its subtypes. Results: After adjusting the effects of confounding variables in the models, the odds of BrCa was significantly lower in the highest intake quartile compared with the lowest quartile for total intake of vitamin B2 (OR = 0.17, 95% CI, 0.07-0.39; Ptrend < 0.001), vitamin B6 (OR = 0.11, 95% CI, 0.05-0.27; Ptrend < 0.001), vitamin B12 (OR = 0.20, 95% CI, 0.09-0.43; Ptrend < 0.001) and folate (OR = 0.09, 95% CI, 0.04-0.21; Ptrend < 0.001). Also, those with the highest quartile of vitamin B6, B12, B2 and folate intake compared with the lowest quartile were less likely to develop estrogen receptor (ER)+ and progesterone receptor (PR)+ subtypes, ER- status, PR- and human epidermal growth factor receptor 2 (HER2)+ subtypes and HER2- status. Conclusion: High intakes of vitamins B2, B6 and folate are associated with reduced odds of BrCa in overall and all ER, PR and HER2 subtypes. Also, high intakes of vitamin B12 reduced the odds of all subtypes of BrCa except ER- subtype.


Assuntos
Neoplasias da Mama , Carbono/química , Ácido Fólico/metabolismo , Vitamina B 12/metabolismo , Vitamina B 6/metabolismo , Vitaminas , Neoplasias da Mama/prevenção & controle , Estudos de Casos e Controles , Dieta , Ácido Fólico/química , Humanos , Receptores Estrogênicos , Fatores de Risco , Vitamina B 12/química , Vitamina B 12/farmacologia , Vitamina B 6/química , Vitamina B 6/farmacologia
8.
Int J Vitam Nutr Res ; 90(1-2): 151-155, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30761942

RESUMO

Vitamin B12 (Cobalamin) deficiency, due to improper internalization of cobalamin, is a metabolic disorder prevalent in impoverished and elderly populations and is associated with megaloblastic anemia and dementia. It has been suggested that mutations in transcobalamin II (TCN2) or gastric intrinsic factor (GIF) proteins can alter their binding efficiency to cobalamin or reduce the ability of their receptors to internalize them. In this case-control study, the correlation between vitamin B12 deficiency and alternative alleles of TCN2 and GIF was investigated in a Jordanian population. One hundred individuals with vitamin B12 deficiency (B12 < 200 mg/mL) were enrolled in our study to evaluate the TCN2 and GIF polymorphisms. The control group (B12 > 200 mg/mL) included 100 individuals. Our results indicated a significant association between the homologous variant of the TCN2 gene (G776G) and vitamin B12 deficiency, and an intermediate phenotype in heterozygous individuals (p < 0.001, OR = 5.6, 95% CI = 2.95 to 10.63). The GIF gene, however, showed no correlation between the A68G variant and vitamin B12 deficiency (p = 0.2). This study expounds the association of TCN2 polymorphism with cobalamin levels in a Jordanian population and highlights the necessity of further studies to elucidate the molecular basis and impact of TCN2 and GIF genes polymorphisms on vitamin B12 deficiency and associated disorders.


Assuntos
Transcobalaminas , Deficiência de Vitamina B 12/sangue , Vitamina B 12/sangue , Idoso , Estudos de Casos e Controles , Humanos , Prevalência , Transcobalaminas/genética , Vitamina B 12/química , Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/complicações , Deficiência de Vitamina B 12/genética
9.
ISME J ; 14(1): 53-66, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31492962

RESUMO

Cobalamin (vitamin B12) is an essential enzyme cofactor for most branches of life. Despite the potential importance of this cofactor for soil microbial communities, the producers and consumers of cobalamin in terrestrial environments are still unknown. Here we provide the first metagenome-based assessment of soil cobalamin-producing bacteria and archaea, quantifying and classifying genes encoding proteins for cobalamin biosynthesis, transport, remodeling, and dependency in 155 soil metagenomes with profile hidden Markov models. We also measured several forms of cobalamin (CN-, Me-, OH-, Ado-B12) and the cobalamin lower ligand (5,6-dimethylbenzimidazole; DMB) in 40 diverse soil samples. Metagenomic analysis revealed that less than 10% of soil bacteria and archaea encode the genetic potential for de novo synthesis of this important enzyme cofactor. Predominant soil cobalamin producers were associated with the Proteobacteria, Actinobacteria, Firmicutes, Nitrospirae, and Thaumarchaeota. In contrast, a much larger proportion of abundant soil genera lacked cobalamin synthesis genes and instead were associated with gene sequences encoding cobalamin transport and cobalamin-dependent enzymes. The enrichment of DMB and corresponding DMB synthesis genes, relative to corrin ring synthesis genes, suggests an important role for cobalamin remodelers in terrestrial habitats. Together, our results indicate that microbial cobalamin production and repair serve as keystone functions that are significantly correlated with microbial community size, diversity, and biogeochemistry of terrestrial ecosystems.


Assuntos
Archaea/metabolismo , Bactérias/metabolismo , Metagenoma , Microbiologia do Solo , Vitamina B 12/metabolismo , Archaea/genética , Bactérias/genética , Ecossistema , Metagenômica , Solo
10.
Proc Natl Acad Sci U S A ; 117(3): 1395-1403, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31848239

RESUMO

Steroid estrogens modulate physiology and development of vertebrates. Conversion of C19 androgens into C18 estrogens is thought to be an irreversible reaction. Here, we report a denitrifying Denitratisoma sp. strain DHT3 capable of catabolizing estrogens or androgens anaerobically. Strain DHT3 genome contains a polycistronic gene cluster, emtABCD, differentially transcribed under estrogen-fed conditions and predicted to encode a cobalamin-dependent methyltransferase system conserved among estrogen-utilizing anaerobes; an emtA-disrupted DHT3 derivative could catabolize androgens but not estrogens. These data, along with the observed androgen production in estrogen-fed strain DHT3 cultures, suggested the occurrence of a cobalamin-dependent estrogen methylation to form androgens. Consistently, the estrogen conversion into androgens in strain DHT3 cell extracts requires methylcobalamin and is inhibited by propyl iodide, a specific inhibitor of cobalamin-dependent enzymes. The identification of the cobalamin-dependent estrogen methylation thus represents an unprecedented metabolic link between cobalamin and steroid metabolism and suggests that retroconversion of estrogens into androgens occurs in the biosphere.


Assuntos
Androgênios/metabolismo , Proteínas de Bactérias/metabolismo , Betaproteobacteria/metabolismo , Estrogênios/metabolismo , Metiltransferases/metabolismo , Vitamina B 12/metabolismo , Proteínas de Bactérias/genética , Betaproteobacteria/enzimologia , Betaproteobacteria/genética , Metiltransferases/genética
11.
Science ; 366(6465): 589-593, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31672889

RESUMO

Itaconate is an immunometabolite with both anti-inflammatory and bactericidal effects. Its coenzyme A (CoA) derivative, itaconyl-CoA, inhibits B12-dependent methylmalonyl-CoA mutase (MCM) by an unknown mechanism. We demonstrate that itaconyl-CoA is a suicide inactivator of human and Mycobacterium tuberculosis MCM, which forms a markedly air-stable biradical adduct with the 5'-deoxyadenosyl moiety of the B12 coenzyme. Termination of the catalytic cycle in this way impairs communication between MCM and its auxiliary repair proteins. Crystallography and spectroscopy of the inhibited enzyme are consistent with a metal-centered cobalt radical ~6 angstroms away from the tertiary carbon-centered radical and suggest a means of controlling radical trajectories during MCM catalysis. Mycobacterial MCM thus joins enzymes in the glyoxylate shunt and the methylcitrate cycle as targets of itaconate in pathogen propionate metabolism.


Assuntos
Coenzima A/metabolismo , Metilmalonil-CoA Mutase/antagonistas & inibidores , Metilmalonil-CoA Mutase/metabolismo , Mycobacterium tuberculosis/enzimologia , Succinatos/metabolismo , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Desoxiadenosinas , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Ligação de Hidrogênio , Macrófagos/metabolismo , Metilmalonil-CoA Mutase/química , Modelos Moleculares , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Propionatos/metabolismo , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Succinatos/farmacologia , Vitamina B 12/metabolismo , Vitamina B 12/farmacologia
12.
Chem Commun (Camb) ; 55(99): 14934-14937, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31774078

RESUMO

Complex carbapenem ß-lactam antibiotics contain diverse C6 alkyl substituents constructed by cobalamin-dependent radical SAM enzymes. TokK installs the C6 isopropyl chain found in asparenomycin. Time-course analyses of TokK and its ortholog ThnK, which forms the C6 ethyl chain of thienamycin, indicate that catalysis occurs through a sequence of discrete, non-processive methyl transfers.


Assuntos
Antibacterianos/biossíntese , Carbapenêmicos/biossíntese , Metionina Adenosiltransferase/metabolismo , Vitamina B 12/metabolismo , Antibacterianos/metabolismo , Carbapenêmicos/metabolismo , Catálise , Metilação
13.
J Food Sci ; 84(10): 2925-2931, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31546283

RESUMO

This study provides phenotypic and molecular analyses of the antibiotic resistance of Ensifer adhaerens strain YX1 (CICC 11008s), a strain that was identified using a polyphasic taxonomy approach. The antibiotic resistance profile of E. adhaerens YX1 was assessed using the Clinical & Laboratory Standards Inst. (CLSI) method. The strain was susceptible to ciprofloxacin, levofloxacin, norfloxacin, ofloxacin, gentamicin, tobramycin, chloramphenicol, tetracycline, imipenem, and ceftazidime, and resistant to kanamycin, streptomycin, fosfomycin, and nitrofurantoin. The antibiotic resistance genes nsfA, nsfB, fosA, aph, and aadA1 were not detected in E. adhaerens YX1 via PCR using gene-specific primers. Subsequently, the genome sequence of E. adhaerens was screened for antibiotic genes. Although no antibiotic resistance genes were identified using the ResFinder database, five genes copies of one resistance gene, adeF, were detected using the Comprehensive Antibiotic Resistance Database (CARD). The results of this study will be useful for understanding the phenotypic and genotypic aspects of E. adhaerens antibiotic resistance. No safety issues were identified for E. adhaerens YX1 in terms of antibiotic resistance. Performing similar studies will be conducive to the safety assessment and control of the use of E. adhaerens in the food and feed industry. PRACTICAL APPLICATION: Few relevant reports are currently available regarding antibiotic resistance assessments or other safety evaluations for Ensifer adhaerens. Because of a lack of relevant information on the safety of this bacterium, including the genetic basis of antibiotic resistance in the production strain, it has not been recommended for use in the "qualified presumption of safety" (QPS) list and subsequent updated lists. The current study shows no safety issue of E. adhaerens YX1 in terms of its antibiotic resistance. These results are important as they provide an initial basis for an understanding of the antibiotic resistance/susceptibility of E. adhaerens YX1 (CICC 11008s), which produces vitamin B12 and is widely used in the food and feed industry.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Rhizobiaceae/efeitos dos fármacos , Vitamina B 12/metabolismo , Ração Animal/microbiologia , Cloranfenicol/farmacologia , Ciprofloxacino/farmacologia , Microbiologia de Alimentos , Testes de Sensibilidade Microbiana , Rhizobiaceae/metabolismo , Tetraciclina/farmacologia
14.
Phys Chem Chem Phys ; 21(37): 20628-20640, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31495862

RESUMO

Methionine synthase (MetH) is a methylcobalamin (MeCbl)-dependent mammalian enzyme which plays a critical role in carrying out the transfer of a methyl group from methyl tetrahydrofolate to homocysteine to generate methionine and tetrahydrofolate. This catalytic cycle proceeds via cleavage of a Co-C bond which is formally heterolytic. This cleavage results in a structural change in the MeCbl cofactor bound to an enzyme. Unlike the native catalysis, upon photoexcitation, the Co-C bond in MeCbl-bound MetH generates the Co(ii)/CH3 radical pairs (RPs). Protein residues of the cap domain, particularly phenylalanine708 (F708) and leucine 715 (L715), which surrounds the upper face of the MeCbl cofactor, inhibit the photolysis of MeCbl by caging the CH3 radical and inducing the geminate recombination of the Co(ii)/CH3 RP. A molecular-level understanding of these effects requires a detailed investigation of the low-lying electronic states. Toward this, we have mutated the F708 residue with alanine (A708) and constructed the potential energy surfaces (PESs) for the low-lying S1 electronic state using a combined quantum mechanics/molecular mechanics (QM/MM) approach. The S1 PESs for the wild-type (WT) and mutant enzymes are the result of crossing of two electronic states, namely metal-to-ligand charge transfer (MLCT) and ligand field (LF) states, indicated by a seam. It is shown that the topologies of the S1 PESs are significantly modulated by introducing a mutation at the F708 position. Specifically, for the WT enzyme, the energy barrier of photoreaction and the energy difference between MLCT and LF minima are markedly higher than those of its mutant counterpart. Moreover, mutation influences the photoactivation of the Co-C bond in enzyme-bound MeCbl by decreasing the rate of geminate recombination and altering the rate of radical pair formation. This theoretical insight was also compared with transient absorption spectroscopic (TAS) studies which are in good agreement with the present findings.


Assuntos
5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/genética , Carbono/química , Cobalto/química , Vitamina B 12/análogos & derivados , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Modelos Químicos , Estrutura Molecular , Mutação/genética , Fotólise , Domínios Proteicos/genética , Vitamina B 12/metabolismo
15.
J Biol Chem ; 294(31): 11726-11727, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375551

RESUMO

Radical S-adenosylmethionine (SAM) (RS) methylases perform methylation reactions at unactivated carbon and phosphorus atoms. RS enzymes typically abstract a hydrogen from their substrates, generating a substrate-centered radical; class B RS methylases catalyze methyl transfer from SAM to cobalamin and then to a substrate-centered carbon or phosphorus radical. Radle et al. now show that Mmp10, an RS enzyme implicated in the methylation of Arg-285 in methyl coenzyme M reductase, binds a methylcobalamin cofactor required for methyl transfer from SAM to a peptide substrate. However, Mmp10 has little sequence homology to known methylases, suggesting this enzyme belongs to a new subclass of B12-dependent RS methylases.


Assuntos
Metaloproteinase 10 da Matriz/metabolismo , S-Adenosilmetionina/metabolismo , Vitamina B 12/metabolismo , Biocatálise , Metilação , Oxirredutases/metabolismo , Peptídeos/metabolismo , S-Adenosilmetionina/química , Especificidade por Substrato , Vitamina B 12/análogos & derivados , Vitamina B 12/química
16.
Biochimie ; 165: 19-31, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31269461

RESUMO

Homocysteine (Hcy) is a key metabolite generated during methionine metabolism. The elevated levels of Hcy in the blood are reffered to as hyperhomocystenimeia (HHcy). The HHcy is caused by impaired metabolism/deficiency of either folate or B12 or defects in Hcy metabolism. Accumulating evidence suggests that HHcy is associated with cardiovascular and brain diseases including atherosclerosis, endothelial injury, and stroke etc. Vitamin B12 (cobalamin; B12) is a water-soluble vitamin essential for two metabolic reactions. It acts as a co-factor for methionine synthase and L-methylmalonyl-CoA mutase. Besides, it is also vital for DNA synthesis and maturation of RBC. Deficiency of B12 is associated with haematological and neurological disorders. Hyperhomocysteinemia (HHcy)-induced toxicity is thought to be mediated by the accumulation of Hcy and its metabolites, homocysteinylated proteins. Cellular protein quality control (PQC) is essential for the maintenance of proteome integrity, and cell viability and its failure contributes to the development of multiple diseases. Chaperones, unfolded protein response (UPR), ubiquitin-proteasome system (UPS), and autophagy are analogous strategies of PQC that maintain cellular proteome integrity. Recently, multiple studies reported that HHcy responsible for perturbation of PQC by reducing chaperone levels, activating UPR, and impairing autophagy. Besides, HHcy also induce cytotoxicity, inflammation, protein aggregation and apoptosis. It has been shown that some of the factors including altered SIRT1-HSF1 axis and irreversible homocysteinylation of proteins are responsible for folate and/or B12 deficiency or HHcy-induced impairment of PQC. Therefore, this review highlights the current understanding of HHcy in the context of cellular PQC and their pathophysiological and clinical consequences, epigenomic changes, therapeutic implications of B12, and chemical chaperones based on cell culture and experimental animal models.


Assuntos
Homocisteína/metabolismo , Hiper-Homocisteinemia/sangue , Vitamina B 12/metabolismo , Animais , Apoptose , Autofagia , Linhagem Celular , Humanos , Camundongos , Chaperonas Moleculares/metabolismo , Agregação Patológica de Proteínas , Processamento de Proteína Pós-Traducional , Ratos , Ubiquitinação , Resposta a Proteínas não Dobradas
17.
Protist ; 170(3): 328-348, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31260945

RESUMO

Diatoms perform an estimated 20% of global photosynthesis, form the base of the marine food web, and sequester carbon into the deep ocean through the biological pump. In some areas of the ocean, diatom growth is limited by the micronutrient cobalamin (vitamin B12), yet the biochemical ramifications of cobalamin limitation are not well understood. In a laboratory setting, we grew the diatom Thalassiosira pseudonana under replete and low cobalamin conditions to elucidate changes in metabolite pools. Using metabolomics, we show that the diatom experienced a metabolic cascade under cobalamin limitation that affected the central methionine cycle, transsulfuration pathway, and composition of osmolyte pools. In T. pseudonana, 5'-methylthioadenosine decreased under low cobalamin conditions, suggesting a disruption in the diatom's polyamine biosynthesis. Furthermore, two acylcarnitines accumulated under low cobalamin, suggesting the limited use of an adenosylcobalamin-dependent enzyme, methylmalonyl CoA mutase. Overall, these changes in metabolite pools yield insight into the metabolic consequences of cobalamin limitation in diatoms and suggest that cobalamin availability may have consequences for microbial interactions that are based on metabolite production by phytoplankton.


Assuntos
Diatomáceas/metabolismo , Metabolômica , Vitamina B 12/metabolismo , Metaboloma
18.
J Appl Biomater Funct Mater ; 17(2): 2280800018817462, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31230497

RESUMO

Alginate scaffold has been used widely for controlled release applications because of its ability to provide three-dimensional supports for formation of a gel matrix. Alginate gel scaffolds for drug delivery matrices were prepared using a fluidic device. N2 gas was used in the fluidic device to generate bubbles in the gel layer. The hydrogel matrices with induced voids were compared with hydrogel matrices without voids. This study attempted to identify the release mechanism of vitamin B12 from the two types of prepared scaffolds, and the data were fitted with different release kinetic models. The results revealed that the alginate scaffold exhibited a controlled release profile and that the corresponding release mechanism followed a first-order kinetic model. Hydrogel scaffolds fabricated with biocompatible polymers using fluidic methods could be promising for controlled drug delivery systems.


Assuntos
Alginatos/química , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Hidrogéis/química , Cinética , Resistência ao Cisalhamento , Vitamina B 12/química , Vitamina B 12/metabolismo
19.
Hum Genet ; 138(7): 703-713, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31139930

RESUMO

Neural tube defects (NTD) result from complex mechanisms between genes, nutrition and environment. The identification of genetic predictors by genome exome sequencing and their influence on genome methylation need further consideration. Gene variants related to 1-CM metabolism (1-CM) could influence the methylation of genes involved in neural tube embryogenesis through impaired synthesis of S-adenosyl methionine. We performed exome sequencing of 6116 genes referenced in OMIM and NTD risk and genome-wide methylation in 23 NTD cases. We replicated the most significant associations in 81 other cases. The analysis of exome sequencing identified one gene of 1-CM, LRP2, and one gene of Sonic Hedgehog (SHH), GLI3, in the 23 NTD cases. The analysis restricted to genes of 1-CM and neural tube embryogenesis identified five gene predictors of 1-CM (LRP2, rs137983840; MMAA, rs148142853; TCN2, rs35838082; FPGS, rs41306702; BHMT, rs763726268) and two of SHH (GLI3, rs35364414; MKS1, rs151023718). We replicated the association of TCN2, BHMT and GLI3 with NTD risk in the 81 cases. We found a significant hemimethylation of CFAP46 that may influence SHH activation in one case, who carried risk alleles in BHMT, LRP2, MMAA and GLI3. In conclusion, we identified new candidate genes and rare variants that highlight an interacting influence of genes involved in SHH and 1-CM in the puzzle of genetic components of NTD risk.


Assuntos
Biomarcadores/metabolismo , Carbono/metabolismo , Exoma , Proteínas Hedgehog/genética , Defeitos do Tubo Neural/genética , Vitamina B 12/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Humanos , Masculino , Defeitos do Tubo Neural/metabolismo , Defeitos do Tubo Neural/patologia , Transdução de Sinais , Sequenciamento Completo do Exoma , Adulto Jovem
20.
Molecules ; 24(9)2019 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-31035627

RESUMO

Nowadays, regenerative medicine has paid special attention to research (in vitro and in vivo) related to bone regeneration, specifically in the treatment of bone fractures or skeletal defects, which is rising worldwide and is continually demanding new developments in the use of stem cells, growth factors, membranes and scaffolds based on novel nanomaterials, and their applications in patients by using advanced tools from molecular biology and tissue engineering. Strontium (Sr) is an element that has been investigated in recent years for its participation in the process of remodeling and bone formation. Based on these antecedents, this is a review about the Strontium Folate (SrFO), a recently developed non-protein based bone-promoting agent with interest in medical and pharmaceutical fields due to its improved features in comparison to current therapies for bone diseases.


Assuntos
Regeneração Óssea , Ácido Fólico/metabolismo , Estrôncio/metabolismo , Tecidos Suporte , Animais , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Polpa Dentária/citologia , Ácido Fólico/química , Humanos , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Estrôncio/química , Engenharia Tecidual , Tecidos Suporte/química , Vitamina B 12/química , Vitamina B 12/metabolismo , Vitamina B 6/química , Vitamina B 6/metabolismo
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