RESUMO
Cofactors are fundamental to the catalytic activity of enzymes. Additionally, because plants are a critical source of several cofactors (i.e., including their vitamin precursors) within the context of human nutrition, there have been several studies aiming to understand the metabolism of coenzymes and vitamins in plants in detail. For example, compelling evidence has been brought forth regarding the role of cofactors in plants; specifically, it is becoming increasingly clear that an adequate supply of cofactors in plants directly affects their development, metabolism, and stress responses. Here, we review the state-of-the-art knowledge on the significance of coenzymes and their precursors with regard to general plant physiology and discuss the emerging functions attributed to them. Furthermore, we discuss how our understanding of the complex relationship between cofactors and plant metabolism can be used for crop improvement.
Assuntos
Coenzimas , Vitaminas , Humanos , Coenzimas/metabolismo , Vitaminas/metabolismo , Plantas/metabolismo , Fenômenos Fisiológicos VegetaisRESUMO
D-Tagatose is a ketohexose, which presents unique properties as a low-calorie functional sweetener possessing a sweet flavor profile similar to D-sucrose and having no aftertaste. Considered a generally recognized as safe (GRAS) substance by FAO/WHO, D-tagatose can be used as an intermediate for the synthesis of other optically active compounds as well as an additive in detergent, cosmetic, and pharmaceutical formulations. This study reports important features for L-arabinose isomerase (EC 5.3.1.4) (L-AI) use in industry. We describe arabinose (araA) gene virulence analysis, gene isolation, sequencing, cloning, and heterologous overexpression of L-AI from the food-grade GRAS bacterium Enterococcus faecium DBFIQ E36 in Escherichia coli and assess biochemical properties of this recombinant enzyme. Recombinant L-AI (rL-AI) was one-step purified to homogeneity by Ni2+-agarose resin affinity chromatography and biochemical characterization revealed low identity with both thermophilic and mesophilic L-AIs but high degree of conservation in residues involved in substrate recognition. Optimal conditions for rL-AI activity were 50 °C, pH 5.5, and 0.3 mM Mn2+, exhibiting a low cofactor concentration requirement and an acidic optimum pH. Half-life at 45 °C and 50 °C were 1427 h and 11 h, respectively, and 21.5 h and 39.5 h at pH 4.5 and 5.6, respectively, showing the high stability of the enzyme in the presence of a metallic cofactor. Bioconversion yield for D-tagatose biosynthesis was 45% at 50 °C after 48 h. These properties highlight the technological potential of E. faecium rL-AI as biocatalyst for D-tagatose production.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Proteínas de Bactérias/metabolismo , Enterococcus faecium/enzimologia , Galactose/metabolismo , Hexoses/biossíntese , Aldose-Cetose Isomerases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cátions Bivalentes , Clonagem Molecular , Coenzimas/metabolismo , Enterococcus faecium/genética , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Manganês/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Most oxidoreductases that use NAD+ or NADP+ to transfer electrons in redox reactions display a strong preference for the cofactor. The catalytic efficiency of peach glucitol dehydrogenase (GolDHase) for NAD+ is 1800-fold higher than that for NADP+. Herein, we combined structural and kinetic data to reverse the cofactor specificity of this enzyme. Using site-saturation mutagenesis, we obtained the D216A mutant, which uses both NAD+ and NADP+, although with different catalytic efficiencies (1000 ± 200 and 170 ± 30 M-1 s-1, respectively). This mutant was used as a template to introduce further mutations by site-directed mutagenesis, using information from the fruit fly NADP-dependent GolDHase. The D216A/V217R/D218S triple mutant displayed a 2-fold higher catalytic efficiency with NADP+ than with NAD+. Overall, our results indicate that the triple mutant has the potential to be used for metabolic and cellular engineering and for cofactor recycling in industrial processes.
Assuntos
Coenzimas/metabolismo , L-Iditol 2-Desidrogenase/metabolismo , NADP/metabolismo , Proteínas de Plantas/metabolismo , Prunus persica/enzimologia , Cinética , L-Iditol 2-Desidrogenase/química , L-Iditol 2-Desidrogenase/genética , Mutagênese Sítio-Dirigida , Proteínas de Plantas/química , Proteínas de Plantas/genéticaRESUMO
Peroxynitrite is a short-lived and reactive biological oxidant formed from the diffusion-controlled reaction of the free radicals superoxide (O2â¢-) and nitric oxide (â¢NO). In this review, we first analyze the biochemical evidence for the formation of peroxynitrite in vivo and the reactions that lead to it. Then, we describe the principal reactions that peroxynitrite undergoes with biological targets and provide kinetic and mechanistic details. In these reactions, peroxynitrite has roles as (1) peroxide, (2) Lewis base, and (3) free radical generator. Physiological levels of CO2 can change the outcome of peroxynitrite reactions. The second part of the review assesses the formation of protein 3-nitrotyrosine (NO2Tyr) by peroxynitrite-dependent and -independent mechanisms, as one of the hallmarks of the actions of â¢NO-derived oxidants in biological systems. Moreover, tyrosine nitration impacts protein structure and function, tyrosine kinase signal transduction cascades and protein turnover. Overall, the review is aimed to provide an integrated biochemical view on the formation and reactions of peroxynitrite under biologically relevant conditions and the impact of this stealthy oxidant and one of its major footprints, protein NO2Tyr, in the disruption of cellular homeostasis.
Assuntos
Ácido Peroxinitroso/metabolismo , Proteínas/metabolismo , Tirosina/metabolismo , Dióxido de Carbono/química , Coenzimas/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Heme/química , Heme/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Cinética , Peroxidases/metabolismo , Ácido Peroxinitroso/química , Proteínas/químicaRESUMO
Leishmania parasites cause a broad spectrum of clinical manifestations in humans and the available clinical treatments are far from satisfactory. Since these pathogens require large amounts of NADPH to maintain intracellular redox homeostasis, oxidoreductases that catalyze the production of NADPH are considered as potential drug targets against these diseases. In the sequenced genomes of most Leishmania spp. two putative malic enzymes (MEs) with an identity of about 55% have been identified. In this work, the ME from L. major (LmjF24.0770, Lmj_ME-70) and its less similar homolog from L. mexicana (LmxM.24.0761, Lmex_ME-61) were cloned and functionally characterized. Both MEs specifically catalyzed NADPH production, but only Lmex_ME-61 was activated by l-aspartate. Unlike the allosterically activated human ME, Lmex_ME-61 exhibited typical hyperbolic curves without any sign of cooperativity in the absence of l-aspartate. Moreover, Lmex_ME-61 and Lmj_ME-70 differ from higher eukaryotic homologs in that they display dimeric instead of tetrameric molecular organization. Homology modeling analysis showed that Lmex_ME-61 and Lmj_ME-70 notably differ in their surface charge distribution; this feature encompasses the coenzyme binding pockets as well. However, in both isozymes, the residues directly involved in the coenzyme binding exhibited a good degree of conservation. Besides, only Lmex_ME-61 and its closest homologs were immunodetected in cell-free extracts from L. mexicana, L. amazonensis and L. braziliensis promastigotes. Our findings provide a first glimpse into the biochemical properties of leishmanial MEs and suggest that MEs could be potentially related to the metabolic differences among the species of Leishmania parasites.
Assuntos
Leishmania major/enzimologia , Leishmania mexicana/enzimologia , Malato Desidrogenase (NADP+)/genética , Malato Desidrogenase (NADP+)/metabolismo , Ácido Aspártico/metabolismo , Sítios de Ligação , Clonagem Molecular , Coenzimas/metabolismo , Biologia Computacional , Expressão Gênica , Leishmania major/genética , Leishmania mexicana/genética , Malato Desidrogenase (NADP+)/química , Modelos Moleculares , NADP/metabolismo , Multimerização ProteicaRESUMO
Human mitochondrial methylmalonyl-CoA mutase (hMCM) is an isomerase that converts methylmalonyl-CoA to succinyl-CoA, a crucial step for the incorporation of some compounds derived from the diet into the central metabolism. hMCM employs highly reactive radicals from its cofactor (adenosylcobalamin, AdoCbl) to perform its reaction. Our previous work demonstrated that hMCM loses activity during catalysis and that the interaction with human MMAA (hMMAA), a GTPase protein, avoided this loss or restored hMCM activity. Even so, the mechanism by which hMMAA exerted these chaperone functions has not been described. In this work report that the formation and accumulation of OH2Cbl, the oxidized form of the AdoCbl cofactor formed during catalysis, is the cause of hMCM inactivation. Additionally, we demonstrate that the complex formation of hMCM/hMMAA decreases the rate of oxidized cofactor formation, protecting the hMCM enzyme. Moreover, an inactive model of hMCM was used to demonstrate that hMMAA is able to remove the damaged cofactor through GTP hydrolysis. Additionally, a modification in the kinetic parameters of hMCM in presence of hMMAA was observed, and for the first time, the in vivo localization of hMMAA and its colocalization with hMCM in human fibroblasts mitochondria were demonstrated.
Assuntos
Coenzimas/metabolismo , Metilmalonil-CoA Mutase/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Fibroblastos/metabolismo , Humanos , Mitocôndrias/metabolismo , Oxirredução , Transporte ProteicoRESUMO
The enzyme glucose-6-phosphate dehydrogenase from Trypanosoma cruzi (TcG6PDH) catalyses the first step of the pentose phosphate pathway (PPP) and is considered a promising target for the discovery of a new drug against Chagas diseases. In the present work, we describe the crystal structure of TcG6PDH obtained in a ternary complex with the substrate ß-d-glucose-6-phosphate (G6P) and the reduced 'catalytic' cofactor NADPH, which reveals the molecular basis of substrate and cofactor recognition. A comparison with the homologous human protein sheds light on differences in the cofactor-binding site that might be explored towards the design of new NADP(+) competitive inhibitors targeting the parasite enzyme.
Assuntos
Doença de Chagas/tratamento farmacológico , Coenzimas/química , Glucosefosfato Desidrogenase/química , Conformação Proteica , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos/genética , Animais , Sítios de Ligação/efeitos dos fármacos , Doença de Chagas/genética , Coenzimas/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Glucose-6-Fosfato/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Humanos , NADP/metabolismo , Via de Pentose Fosfato/genética , Especificidade por Substrato , Trypanosoma cruzi/patogenicidadeRESUMO
Three novel D-xylose-fermenting yeast species of Spathaspora clade were recovered from rotting wood in regions of the Atlantic Rainforest ecosystem in Brazil. Differentiation of new species was based on analyses of the gene encoding the D1/D2 sequences of large subunit of rRNA and on 642 conserved, single-copy, orthologous genes from genome sequence assemblies from the newly described species and 15 closely-related Debaryomycetaceae/Metschnikowiaceae species. Spathaspora girioi sp. nov. produced unconjugated asci with a single elongated ascospore with curved ends; ascospore formation was not observed for the other two species. The three novel species ferment D-xylose with different efficiencies. Spathaspora hagerdaliae sp. nov. and Sp. girioi sp. nov. showed xylose reductase (XR) activity strictly dependent on NADPH, whereas Sp. gorwiae sp. nov. had XR activity that used both NADH and NADPH as co-factors. The genes that encode enzymes involved in D-xylose metabolism (XR, xylitol dehydrogenase and xylulokinase) were also identified for these novel species. The type strains are Sp. girioi sp. nov. UFMG-CM-Y302(T) (=CBS 13476), Sp. hagerdaliae f.a., sp. nov. UFMG-CM-Y303(T) (=CBS 13475) and Sp. gorwiae f.a., sp. nov. UFMG-CM-Y312(T) (=CBS 13472).
Assuntos
Fermentação , Genoma Fúngico , Genômica , Saccharomycetales/classificação , Saccharomycetales/metabolismo , Xilose/metabolismo , Brasil , Análise por Conglomerados , Coenzimas/metabolismo , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , NAD/metabolismo , NADP/metabolismo , Filogenia , RNA Ribossômico/genética , Saccharomycetales/genética , Saccharomycetales/isolamento & purificação , Análise de Sequência de DNA , Esporos Fúngicos/citologia , Madeira/microbiologiaRESUMO
In bacteria, proteins containing GGDEF domains are involved in production of the second messenger c-di-GMP. Here we report that the cdgA gene encoding diguanylate cyclase A (CdgA) is involved in biofilm formation and exopolysaccharide (EPS) production in Azospirillum brasilense Sp7. Biofilm quantification using crystal violet staining revealed that inactivation of cdgA decreased biofilm formation. In addition, confocal laser scanning microscopy analysis of green-fluorescent protein-labeled bacteria showed that, during static growth, the biofilms had differential levels of development: bacteria harboring a cdgA mutation exhibited biofilms with considerably reduced thickness compared with those of the wild-type Sp7 strain. Moreover, DNA-specific staining and treatment with DNase I, and epifluorescence studies demonstrated that extracellular DNA and EPS are components of the biofilm matrix in Azospirillum. After expression and purification of the CdgA protein, diguanylate cyclase activity was detected. The enzymatic activity of CdgA-producing cyclic c-di-GMP was determined using GTP as a substrate and flavin adenine dinucleotide (FAD(+)) and Mg(2)(+) as cofactors. Together, our results revealed that A. brasilense possesses a functional c-di-GMP biosynthesis pathway.
Assuntos
Azospirillum brasilense/enzimologia , Azospirillum brasilense/fisiologia , Biofilmes/crescimento & desenvolvimento , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Polissacarídeos Bacterianos/biossíntese , Azospirillum brasilense/genética , Técnicas Bacteriológicas , Coenzimas/metabolismo , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/isolamento & purificação , Flavina-Adenina Dinucleotídeo/metabolismo , Guanosina Trifosfato/metabolismo , Magnésio/metabolismo , Microscopia Confocal , Fósforo-Oxigênio Liases/isolamento & purificação , Coloração e RotulagemRESUMO
A healthy physiology depends on a plethora of complex interdependent biochemical reactions. In order for these reactions to occur suitably, the enzymes and cofactors that regulate their flow must be present in the proper balance. The term metabolic correction is used to describe a biochemical-physiological process that improves cellular biochemistry as a means to an individual's achieving metabolic or physiological optimization. Part 2 discusses how metabolic correction, through the increase of cofactors, can supply unmet enzyme needs and compensate for nutritional deficiencies induced by improper nutritional intake or by the increased demand for nutrients caused by genetics, health conditions, medications, or physical or environmental stressors. Nutrient insufficiencies are causing an increase in morbidity and mortality, at great cost to our society. In summary, metabolic correction improves enzymatic function and satisfies the increasing demand for nutrients. Metabolic correction can have a significant impact on the reduction of morbidity and mortality and their financial cost to our society and contribute to improving health and well-being.