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1.
Proc Natl Acad Sci U S A ; 117(20): 10818-10824, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32371483

RESUMO

Recent advances in neutron crystallographic studies have provided structural bases for quantum behaviors of protons observed in enzymatic reactions. Thus, we resolved the neutron crystal structure of a bacterial copper (Cu) amine oxidase (CAO), which contains a prosthetic Cu ion and a protein-derived redox cofactor, topa quinone (TPQ). We solved hitherto unknown structures of the active site, including a keto/enolate equilibrium of the cofactor with a nonplanar quinone ring, unusual proton sharing between the cofactor and the catalytic base, and metal-induced deprotonation of a histidine residue that coordinates to the Cu. Our findings show a refined active-site structure that gives detailed information on the protonation state of dissociable groups, such as the quinone cofactor, which are critical for catalytic reactions.


Assuntos
Amina Oxidase (contendo Cobre)/química , Proteínas de Bactérias/química , Quinonas/química , Domínio Catalítico , Coenzimas/química , Difração de Nêutrons , Prótons
2.
Artigo em Inglês | MEDLINE | ID: mdl-32161212

RESUMO

In order to harness the functionality of metals, nature has evolved over billions of years to utilize metalloproteins as key components in numerous cellular processes. Despite this, transition metals such as ruthenium, palladium, iridium, and gold are largely absent from naturally occurring metalloproteins, likely due to their scarcity as precious metals. To mimic the evolutionary process of nature, the field of artificial metalloenzymes (ArMs) was born as a way to benefit from the unique chemoselectivity and orthogonality of transition metals in a biological setting. In its current state, numerous examples have successfully incorporated transition metals into a variety of protein scaffolds. Using these ArMs, many examples of new-to-nature reactions have been carried out, some of which have shown substantial biocompatibility. Given the rapid rate at which this field is growing, this review aims to highlight some important studies that have begun to take the next step within this field; namely the development of ArM-centered drug therapies or biotechnological tools.


Assuntos
Materiais Biomiméticos/química , Coenzimas/química , Metaloproteínas/química , Metais/química , Biocatálise , Técnicas Biossensoriais , Modelos Moleculares , Conformação Proteica , Engenharia de Proteínas , Estereoisomerismo
3.
Nat Commun ; 11(1): 620, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001697

RESUMO

Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5'-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds.


Assuntos
Coenzimas/química , Cristalização , IMP Desidrogenase/química , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , Guanosina Monofosfato , Modelos Moleculares , Conformação Proteica , Células Sf9 , Trypanosoma brucei brucei/genética
4.
J Biosci Bioeng ; 129(5): 552-557, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31983661

RESUMO

The gut bacterium Gordonibacter urolithinfaciens DSM 27213 metabolizes ellagic acid into three polyphenol compounds, namely, urolithin M5, urolithin M6, and urolithin C, which are collectively called urolithin. The key reactions of this metabolic pathway are the dehydroxylation of the phenolic hydroxy group, i.e., conversion of urolithin M5 to urolithin M6, and successive conversion of urolithin M6 to urolithin C. By testing the effects of various electron-transferring compounds on the dehydroxylation reactions, methylviologen was found to effectively support the dehydroxylation catalyzed by the cell free extracts. The urolithin dehydroxylating enzymes were found in the soluble fraction of the cell free extracts. The urolithin dehydroxylation was found to be coupled with reduction of dicationic methylviologen to a cation radical form catalyzed by enzymes with hydrogen as an electron donor, which was also found with the soluble fraction. Further investigation of the reaction in the presence of natural cofactors with or without methylviologen and hydrogen revealed the involvement of NADPH and FAD in the electron transportation systems of the urolithin dehydroxylation.


Assuntos
Actinobacteria/enzimologia , Proteínas de Bactérias/metabolismo , Coenzimas/metabolismo , Taninos Hidrolisáveis/metabolismo , Actinobacteria/química , Actinobacteria/metabolismo , Proteínas de Bactérias/genética , Elétrons , Taninos Hidrolisáveis/química , Hidroxilação
5.
Appl Biochem Biotechnol ; 190(1): 18-29, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31301008

RESUMO

NAD(P)H-dependent enzymes are ideal biocatalysts for the industrial production of chiral compounds, such as chiral alcohols, chiral amino acids, and chiral amines; however, efficient strategies for the regeneration of coenzyme are expected as costly of the coenzymes. Herein, a solvent-tolerant isopropanol dehydrogenase (IDH) showing lower similarity (37%) with other proteins was obtained and characterized. The enzyme exhibits high catalysis ability of its substrates methanol, ethanol, ethylene glycol, glycerol, isopropanol, n-butanol, isobutanol, and acetone. And it has good adaptability in organic solvents (isopropanol, acetonitrile, acetone, and acetophenone). Interaction force and the corresponding amino acid residues between IDH and NAD+ or NADP+ were parsed by docking. The wide substrate spectrum, excellent organic solvent tolerance, and good biocatalytic activity make the excavated enzyme a promising biocatalyst for the production of chiral compounds industrially and the construction of coenzyme regeneration systems in aqueous organic phase or organic phase.


Assuntos
Oxirredutases do Álcool/metabolismo , Coenzimas/metabolismo , Solventes/metabolismo , Oxirredutases do Álcool/genética , Sítios de Ligação , Clonagem Molecular , Cinética , Simulação de Acoplamento Molecular , NAD/metabolismo , NADP/metabolismo , Compostos Orgânicos/metabolismo , Especificidade por Substrato
6.
Inorg Chem ; 59(1): 214-225, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31814403

RESUMO

Formate dehydrogenase (FDH) enzymes are versatile catalysts for CO2 conversion. The FDH from Rhodobacter capsulatus contains a molybdenum cofactor with the dithiolene functions of two pyranopterin guanine dinucleotide molecules, a conserved cysteine, and a sulfido group bound at Mo(VI). In this study, we focused on metal oxidation state and coordination changes in response to exposure to O2, inhibitory anions, and redox agents using X-ray absorption spectroscopy (XAS) at the Mo K-edge. Differences in the oxidative modification of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor relative to samples prepared aerobically without inhibitor, such as variations in the relative numbers of sulfido (Mo═S) and oxo (Mo═O) bonds, were observed in the presence of azide (N3-) or cyanate (OCN-). Azide provided best protection against O2, resulting in a quantitatively sulfurated cofactor with a displaced cysteine ligand and optimized formate oxidation activity. Replacement of the cysteine ligand by a formate (HCO2-) ligand at the molybdenum in active enzyme is compatible with our XAS data. Cyanide (CN-) inactivated the enzyme by replacing the sulfido ligand at Mo(VI) with an oxo ligand. Evidence that the sulfido group may become protonated upon molybdenum reduction was obtained. Our results emphasize the role of coordination flexibility at the molybdenum center during inhibitory and catalytic processes of FDH enzymes.


Assuntos
Coenzimas/química , Formiato Desidrogenases/química , Metaloproteínas/química , Pteridinas/química , Rhodobacter capsulatus/enzimologia , Ânions/química , Ânions/metabolismo , Sítios de Ligação , Coenzimas/metabolismo , Formiato Desidrogenases/isolamento & purificação , Formiato Desidrogenases/metabolismo , Metaloproteínas/metabolismo , Oxirredução , Pteridinas/metabolismo , Espectroscopia por Absorção de Raios X
7.
Biochim Biophys Acta Proteins Proteom ; 1868(2): 140322, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31740415

RESUMO

Biocatalysis, the use of enzymes in chemical transformations, is an important green chemistry tool. Cascade reactions combine different enzyme activities in a sequential set of reactions. Cascades can occur within a living (usually bacterial) cell; in vitro in 'one pot' systems where the desired enzymes are mixed together to carry out the multi-enzyme reaction; or using microfluidic systems. Microfluidics offers particular advantages when the product of the reaction inhibits the enzyme(s). In vitro systems allow variation of different enzyme concentrations to optimise the metabolic 'flux', and the addition of enzyme cofactors as required. Cascades including cofactor recycling systems and modelling approaches are being developed to optimise cascades for wider industrial scale use. Two industrially important enzymes, transaminases and carboxylic acid reductases are used as examples regarding their applications in cascade reactions with other enzyme classes to obtain important synthons of pharmaceutical interest.


Assuntos
Oxirredutases/metabolismo , Transaminases/metabolismo , Biocatálise , Coenzimas/metabolismo , Química Verde , Cinética , Microfluídica/métodos , Oxirredutases/antagonistas & inibidores , Transaminases/antagonistas & inibidores
8.
Colloids Surf B Biointerfaces ; 185: 110624, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31711735

RESUMO

This manuscript presents a novel bioanalytical approach for the selective ratiometric fluorescent sensing of enzymatic activity of the alkaline phosphatase (ALP) in the biological samples. The probe was designed by conjugating the pyridoxal 5'-phosphate (PLP) over the surface of bovine serum albumin (BSA) stabilized CdS quantum dots (QDs) through the interaction of free amine present in BSA with the aldehyde group of PLP. The conjugation of PLP quenched the emission of QDs. Upon addition of the ALP, the emission of QDs was restored due to the dephosphorylation and the conversion of the functionalized PLP in to pyridoxal. With this probe, the ALP activity can be detected down to 0.05 U/L and also successfully applied for the detection of ALP activity in biological samples such as human serum and plasma.


Assuntos
Fosfatase Alcalina/análise , Compostos de Cádmio/química , Coenzimas/metabolismo , Pontos Quânticos/química , Soroalbumina Bovina/metabolismo , Sulfetos/química , Vitamina B 6/metabolismo , Fosfatase Alcalina/sangue , Animais , Bovinos , Humanos , Fosfato de Piridoxal/metabolismo , Espectrometria de Fluorescência
9.
Adv Exp Med Biol ; 1203: 113-132, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31811632

RESUMO

The RNA exosome is a highly conserved ribonuclease endowed with 3'-5' exonuclease and endonuclease activities. The multisubunit complex resides in both the nucleus and the cytoplasm, with varying compositions and activities between the two compartments. While the cytoplasmic exosome functions mostly in mRNA quality control pathways, the nuclear RNA exosome partakes in the 3'-end processing and complete decay of a wide variety of substrates, including virtually all types of noncoding (nc) RNAs. To handle these diverse tasks, the nuclear exosome engages with dedicated cofactors, some of which serve as activators by stimulating decay through oligoA addition and/or RNA helicase activities or, as adaptors, by recruiting RNA substrates through their RNA-binding capacities. Most nuclear exosome cofactors contain the essential RNA helicase Mtr4 (MTR4 in humans). However, apart from Mtr4, nuclear exosome cofactors have undergone significant evolutionary divergence. Here, we summarize biochemical and functional knowledge about the nuclear exosome and exemplify its cofactor variety by discussing the best understood model organisms-the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe, and human cells.


Assuntos
Coenzimas , Complexo Multienzimático de Ribonucleases do Exossomo , RNA Nuclear , Coenzimas/metabolismo , RNA Helicases DEAD-box/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Humanos , RNA/metabolismo , RNA Nuclear/metabolismo , RNA não Traduzido/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
Int J Mol Sci ; 20(23)2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31817029

RESUMO

A triphenylmethane reductase derived from Citrobacter sp. KCTC 18061P was coupled with a glucose 1-dehydrogenase from Bacillus sp. ZJ to construct a cofactor self-sufficient bienzyme biocatalytic system for dye decolorization. Fed-batch experiments showed that the system is robust to maintain its activity after 15 cycles without the addition of any expensive exogenous NADH. Subsequently, three different machine learning approaches, including multiple linear regression (MLR), random forest (RF), and artificial neural network (ANN), were employed to explore the response of decolorization efficiency to the variables of the bienzyme system. Statistical parameters of these models suggested that a three-layered ANN model with six hidden neurons was capable of predicting the dye decolorization efficiency with the best accuracy, compared with the models constructed by MLR and RF. Weights analysis of the ANN model showed that the ratio between two enzymes appeared to be the most influential factor, with a relative importance of 54.99% during the decolorization process. The modeling results confirmed that the neural networks could effectively reproduce experimental data and predict the behavior of the decolorization process, especially for complex systems containing multienzymes.


Assuntos
Biocatálise , Coenzimas/metabolismo , Corantes/metabolismo , Modelos Teóricos , Oxirredutases/metabolismo , Algoritmos , Biodegradação Ambiental , Cor , Modelos Lineares , Redes Neurais de Computação , Especificidade por Substrato , Compostos de Tritil/metabolismo
11.
Proc Natl Acad Sci U S A ; 116(51): 25583-25590, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31776258

RESUMO

Methylotrophy, the ability of microorganisms to grow on reduced one-carbon substrates such as methane or methanol, is a feature of various bacterial species. The prevailing oxidation pathway depends on tetrahydromethanopterin (H4MPT) and methylofuran (MYFR), an analog of methanofuran from methanogenic archaea. Formyltransferase/hydrolase complex (Fhc) generates formate from formyl-H4MPT in two consecutive reactions where MYFR acts as a carrier of one-carbon units. Recently, we chemically characterized MYFR from the model methylotroph Methylorubrum extorquens and identified an unusually long polyglutamate side chain of up to 24 glutamates. Here, we report on the crystal structure of Fhc to investigate the function of the polyglutamate side chain in MYFR and the relatedness of the enzyme complex with the orthologous enzymes in archaea. We identified MYFR as a prosthetic group that is tightly, but noncovalently, bound to Fhc. Surprisingly, the structure of Fhc together with MYFR revealed that the polyglutamate side chain of MYFR is branched and contains glutamates with amide bonds at both their α- and γ-carboxyl groups. This negatively charged and branched polyglutamate side chain interacts with a cluster of conserved positively charged residues of Fhc, allowing for strong interactions. The MYFR binding site is located equidistantly from the active site of the formyltransferase (FhcD) and metallo-hydrolase (FhcA). The polyglutamate serves therefore an additional function as a swinging linker to shuttle the one-carbon carrying amine between the two active sites, thereby likely increasing overall catalysis while decreasing the need for high intracellular MYFR concentrations.


Assuntos
Proteínas de Bactérias , Furanos , Hidroximetil e Formil Transferases , Metano , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Coenzimas/química , Coenzimas/metabolismo , Cristalografia , Formiatos/química , Formiatos/metabolismo , Furanos/química , Furanos/metabolismo , Hidroximetil e Formil Transferases/química , Hidroximetil e Formil Transferases/genética , Hidroximetil e Formil Transferases/metabolismo , Metano/química , Metano/metabolismo , Metanol/química , Metanol/metabolismo , Methylobacterium extorquens/enzimologia , Methylobacterium extorquens/genética , Ácido Poliglutâmico/química , Ácido Poliglutâmico/metabolismo
12.
Sheng Wu Gong Cheng Xue Bao ; 35(10): 1942-1954, 2019 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-31668040

RESUMO

The chemical manufacturing industry that uses fossil resources as raw materials, consumes non-renewable resources and also causes damage to the ecological environment, stimulating the development of bio-manufacturing with renewable resources as raw materials. Unlike traditional chemical manufacturing, bio-manufacturing uses cells as a "production workshop", and each process in the "workshop" is catalyzed by enzymes. In addition to mild reaction conditions, the "cell factory" has strong plasticity, and can be used to synthesize various target chemicals according to demand adjustment or reconstitution of metabolic pathways. The design process of the "cell factory" follows the following guidelines: 1) Construct an optimal synthetic route from raw materials to products; 2) Balance the metabolic flux of each reaction in the metabolic pathway, so that the metabolic flux of this pathway is much higher than the primary metabolism of the cells; 3) Precursor supply in the pathway should be sufficient, and adjust multiple precursors supply ratio as needed; 4) enzymatic reactions often involve the participation of various cofactors, smooth metabolic pathways need to balance or regenerate various cofactors; 5) Through genetic modification or process improvement to remove metabolic intermediates and products feedback inhibition to achieve higher yields.


Assuntos
Biotecnologia , Células/metabolismo , Engenharia Metabólica , Redes e Vias Metabólicas , Coenzimas/metabolismo , Redes e Vias Metabólicas/genética
13.
Enzymes ; 45: 225-256, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627878

RESUMO

Despite their relative simplicity, iron-sulfur clusters have been omnipresent as cofactors in myriad cellular processes such as oxidative phosphorylation and other respiratory pathways. Recent research advances confirm the presence of different clusters in enzymes involved in nucleic acid metabolism. Iron-sulfur clusters can therefore be considered hallmarks of cellular metabolism. Helicases, nucleases, glycosylases, DNA polymerases and transcription factors, among others, incorporate various types of clusters that serve differing roles. In this chapter, we review our current understanding of the identity and functions of iron-sulfur clusters in DNA and RNA metabolizing enzymes, highlighting their importance as regulators of cellular function.


Assuntos
Coenzimas/metabolismo , Proteínas com Ferro-Enxofre/química , Proteínas com Ferro-Enxofre/metabolismo , Ácidos Nucleicos/metabolismo , Coenzimas/química , DNA Glicosilases/química , DNA Glicosilases/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleases/química , Desoxirribonucleases/metabolismo
14.
Enzyme Microb Technol ; 131: 109433, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31615666

RESUMO

Azoreductase from Chromobacterium violaceum was characterized biophysically using experimental and computational tools. The in-silico docking and cross-linking experiments using glutaraldehyde suggest dimeric nature of the enzyme. The enzyme structure was modelled and also studied using circular dichroism (CD) spectroscopy which suggests 40% α- helix, 30% ß- sheet and 30% random coils. In the modelled structure of the azoreductase, the cofactor flavin mononucleotide (FMN) binding energy was -3.8 kJ/mol. The binding of FMN affects the azoreductase-cofactor complex stability. The stability-folding studies indicate that the cofactor, FMN is required for folding, stability and activity. Overall, the data provides interesting insight into stability and biophysical parameters of the azoreductase protein.


Assuntos
Chromobacterium/enzimologia , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/metabolismo , Dobramento de Proteína , Dicroísmo Circular , Coenzimas/metabolismo , Mononucleotídeo de Flavina/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Estabilidade Proteica
15.
Arch Biochem Biophys ; 676: 108139, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31622586

RESUMO

Kanosamine is an aminosugar antibiotic, and component of complex antibiotics such as kanamycin. The biosynthesis of kanosamine varies among different bacteria; best known is a pathway starting from UDP-glucose, but Bacillus subtilis can produce kanosamine in a three-step pathway from glucose 6-phosphate. A set of genes proposed to encode a kanosamine pathway has previously been identified within the zwittermicin A gene cluster of Bacillus cereus UW85. These genes, designated kabABC, are similar to the B. subtilis kanosamine pathway genes (ntdABC), but have never been studied experimentally. We have expressed each of the kab genes, and studied the in vitro substrate scope and reaction rates and kinetic mechanisms of all three enzymes. The kab genes encode enzymes that catalyze a route similar to that found in B. subtilis from glucose 6-phosphate to kanosamine, passing through an unusual and unstable 3-keto intermediate. Kinetic studies show the first step in the pathway, the KabC-catalyzed oxidation of glucose 6-phosphate at carbon-3, is very slow relative to the subsequent KabA-catalyzed aminotransferase and KabB-catalyzed phosphatase reactions. KabC differs from its homolog, NtdC, in that it is NADP- rather than NAD-dependent. The KabA kinetic study is the first such report for a kanosamine 6-phosphate aminotransferase, revealing an extremely efficient PLP-dependent reaction. These results show that this kanosamine biosynthesis pathway occurs in more than one organism, and that the reactions are tuned in order to avoid any accumulation of the unstable intermediate.


Assuntos
Bacillus cereus/metabolismo , Proteínas de Bactérias/metabolismo , Bacillus cereus/enzimologia , Biocatálise , Coenzimas/metabolismo , Glucosamina/biossíntese , Cinética , Niacinamida/metabolismo
17.
Biochemistry (Mosc) ; 84(8): 829-850, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31522667

RESUMO

Thiamine (vitamin B1) is a precursor of the well-known coenzyme of central metabolic pathways thiamine diphosphate (ThDP). Highly intense glucose oxidation in the brain requires ThDP-dependent enzymes, which determines the critical significance of thiamine for neuronal functions. However, thiamine can also act through the non-coenzyme mechanisms. The well-known facilitation of acetylcholinergic neurotransmission upon the thiamine and acetylcholine co-release into the synaptic cleft has been supported by the discovery of thiamine triphosphate (ThTP)-dependent phosphorylation of the acetylcholine receptor-associated protein rapsyn, and thiamine interaction with the TAS2R1 receptor, resulting in the activation of synaptic ion currents. The non-coenzyme regulatory binding of thiamine compounds has been demonstrated for the transcriptional regulator p53, poly(ADP-ribose) polymerase, prion protein PRNP, and a number of key metabolic enzymes that do not use ThDP as a coenzyme. The accumulated data indicate that the molecular mechanisms of the neurotropic action of thiamine are far broader than it has been originally believed, and closely linked to the metabolism of thiamine and its derivatives in animals. The significance of this topic has been illustrated by the recently established competition between thiamine and the antidiabetic drug metformin for common transporters, which can be the reason for the thiamine deficiency underlying metformin side effects. Here, we also discuss the medical implications of the research on thiamine, including the role of thiaminases in thiamine reutilization and biosynthesis of thiamine antagonists; molecular mechanisms of action of natural and synthetic thiamine antagonists, and biotransformation of pharmacological forms of thiamine. Given the wide medical application of thiamine and its synthetic forms, these aspects are of high importance for medicine and pharmacology, including the therapy of neurodegenerative diseases.


Assuntos
Hipoglicemiantes/metabolismo , Metformina/metabolismo , Tiamina/análogos & derivados , Tiamina/metabolismo , Complexo Vitamínico B/metabolismo , Animais , Encéfalo/metabolismo , Coenzimas , Humanos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/efeitos adversos , Metformina/administração & dosagem , Metformina/efeitos adversos , Camundongos , Fosforilação , Transporte Proteico/fisiologia , Ratos , Tiamina/efeitos adversos , Tiamina/farmacologia , Deficiência de Tiamina/etiologia , Deficiência de Tiamina/prevenção & controle , Tiamina Pirofosfato/metabolismo , Complexo Vitamínico B/efeitos adversos , Complexo Vitamínico B/farmacologia
18.
Methods Mol Biol ; 2037: 97-110, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31463841

RESUMO

Cellular coenzymes including coenzyme A (CoA), acetyl coenzyme A (acetyl-CoA), coenzymes of redox reactions and of energy, and antioxidants mediate biochemical reactions fundamental to the functioning of all living cells. The redox coenzymes include NAD+ (oxidized nicotinamide adenine dinucleotide), NADH (reduced nicotinamide adenine dinucleotide), NADP+ (oxidized nicotinamide adenine dinucleotide phosphate), and NADPH (reduced nicotinamide adenine dinucleotide phosphate); the energy coenzymes include ATP (adenosine triphosphate), ADP (adenosine diphosphate), and AMP (adenosine monophosphate); and the antioxidants include GSSG (oxidized glutathione) and GSH (reduced glutathione). Their measurement is important to better understand cellular metabolism. Recent advances have pushed the limit of metabolite quantitation using NMR methods to an unprecedented level, which offer a new avenue for analysis of the coenzymes and antioxidants. Unlike the conventional enzyme assays, which need separate protocols for analysis, a simple 1D 1H NMR experiment enables analysis of all these molecular species in one step. In this chapter, we describe protocols for their identification and quantitation in tissue and whole blood using NMR spectroscopy.


Assuntos
Antioxidantes/análise , Antioxidantes/metabolismo , Coenzimas/sangue , Coenzimas/metabolismo , Metabolômica/métodos , Espectroscopia de Prótons por Ressonância Magnética/métodos , Humanos
19.
Nature ; 571(7766): 515-520, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31341297

RESUMO

The mitochondrial ADP/ATP carrier (AAC) is a major transport protein of the inner mitochondrial membrane. It exchanges mitochondrial ATP for cytosolic ADP and controls cellular production of ATP. In addition, it has been proposed that AAC mediates mitochondrial uncoupling, but it has proven difficult to demonstrate this function or to elucidate its mechanisms. Here we record AAC currents directly from inner mitochondrial membranes from various mouse tissues and identify two distinct transport modes: ADP/ATP exchange and H+ transport. The AAC-mediated H+ current requires free fatty acids and resembles the H+ leak via the thermogenic uncoupling protein 1 found in brown fat. The ADP/ATP exchange via AAC negatively regulates the H+ leak, but does not completely inhibit it. This suggests that the H+ leak and mitochondrial uncoupling could be dynamically controlled by cellular ATP demand and the rate of ADP/ATP exchange. By mediating two distinct transport modes, ADP/ATP exchange and H+ leak, AAC connects coupled (ATP production) and uncoupled (thermogenesis) energy conversion in mitochondria.


Assuntos
Mitocôndrias/metabolismo , Translocases Mitocondriais de ADP e ATP/metabolismo , Prótons , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Coenzimas/metabolismo , Ácidos Graxos/metabolismo , Transporte de Íons , Masculino , Camundongos , Consumo de Oxigênio
20.
Phys Chem Chem Phys ; 21(28): 15747-15759, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31276128

RESUMO

The catalytic cofactor of the most common form of nitrogenase contains seven irons and one molybdenum bound together by sulfide bonds. Surprisingly, a central carbide has been demonstrated by experiments. Another noteworthy structural component is a large homocitrate ligand. In recent theoretical studies it has been shown that the central carbide is needed as a place for the incoming protons that are necessary parts of a reduction process. It has also been shown that a role for the homocitrate ligand could be that it may be rotated to release one bond to molybdenum. In the present study, the carbide protonation steps are reinvestigated with similar results to those reported before. The actual activation of N2 in the E4 state is an extremely complicated process. It has been found experimentally that two hydrides should leave as H2, in a reductive elimination process, to allow N2 activation in E4 in an easily reversible step. It is here suggested that after H2 is released, it is necessary for the metal cofactor to get rid of one proton. This is achieved by protonating the homocitrate and then rotating it to release one of the bonds to Mo. After this rotation, N2 can bind. In the E5 step, the homocitrate is rotated back to its original position and remains that way until the end of the catalytic process. The N2 protonation steps are energetically easy. Since a protonated carbide has never been observed experimentally, it is necessary to also have a mechanism for deprotonating the carbon at the end of the catalytic cycles. Such a mechanism is suggested here.


Assuntos
Nitrogenase/metabolismo , Carbono/química , Coenzimas/química , Ativação Enzimática , Metais/química , Nitrogenase/química , Prótons
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