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1.
Talanta ; 236: 122840, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635230

RESUMO

Ultrasensitive detection of biomarkers at an early stage is generally limited by external influence factors such as high reaction temperature, complex operations, and sophisticated instruments. Here, we circumvent these problems by using nicotinamide adenine dinucleotide (NAD+) to control electroinitiated reversible addition fragmentation chain transfer (electro-RAFT) polymerization for biosensing that enables the detection of a few molecules of target DNA. In this coenzyme-catalyzed electro-RAFT polymerization, numerous ferrocenylmethyl methacrylate (FCMMA) as monomer with electrochemistry signal were linked to the biomarker on Au electrode. Afterwards, a strong oxidation peak appears at the potential of about 0.3 V that represents a typical oxidation potential of FCMMA. The sensitivity of this methodology was presented by detecting DNA from 10-1 to 104 fM concentration and detection limit (LOD) being down to 4.39 aM in 10 µL samples. This is lower by factors than detection limits of most other ultra-sensitive electrochemical DNA assays.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Catálise , Coenzimas , DNA , Polimerização
2.
J Med Case Rep ; 15(1): 441, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34465376

RESUMO

BACKGROUND: Very long-chain acyl-coenzyme-A dehydrogenase deficiency is a rare, severe life-threatening metabolic disorder of mitochondrial fatty acid oxidation, caused by mutations in ACADVL gene. Here we present a genetically confirmed case of a South Asian baby girl with severe, early-onset form of very long-chain acyl-coenzyme-A dehydrogenase deficiency due to a novel mutation in ACADVL gene. CASE PRESENTATION: Index case was the second baby girl of second-degree consanguineous South Asian parents. She had an uncomplicated antenatal period and was born by spontaneous vaginal delivery at term with a birth weight of 2910 g. She had been noted to have fair skin complexion, hypotonia, and 3 cm firm hepatomegaly. Since birth, the baby developed grunting, poor feeding, and recurrent episodes of symptomatic hypoglycemia and convulsions with multiple semiology. Her septic screening and urine ketone bodies were negative. The baby had high anion gap metabolic acidosis and elevated transaminases and serum creatine phosphokinase levels. Echocardiogram at 4 months revealed bilateral ventricular hypertrophy. Acylcarnitine profile revealed elevated concentrations of tetradecanoylcarnitine (C14), tetradecanoylcarnitine C14:1, and C14:1/C16. Unfortunately, the baby died due to intercurrent respiratory illness at 4 months of age. Sequence analysis of ACADVL gene in perimortem blood sample revealed homozygous frame shift novel variant NM_001270447.1, c.711_712del p.(Phe237Leufs*38), which confirmed the diagnosis of very long-chain acyl-coenzyme-A dehydrogenase deficiency. CONCLUSIONS: This case demonstrates the importance of early diagnosis and management of very long-chain acyl-coenzyme-A dehydrogenase deficiency in improving the outcome of the patients. Implementation of newborn screening using tandem mass spectrometry in Sri Lanka will be beneficial to reduce the morbidity and mortality of treatable disorders of inborn errors.


Assuntos
Erros Inatos do Metabolismo Lipídico , Acil-CoA Desidrogenase de Cadeia Longa/genética , Criança , Coenzimas , Síndrome Congênita de Insuficiência da Medula Óssea , Feminino , Humanos , Lactente , Recém-Nascido , Erros Inatos do Metabolismo Lipídico/genética , Doenças Mitocondriais , Doenças Musculares , Mutação , Gravidez
3.
BMJ Case Rep ; 14(9)2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34544705

RESUMO

Immune-mediated necrotising myopathy is a subtype of idiopathic inflammatory myopathy characterised by muscle fibre necrosis without significant inflammatory infiltrate. Anti-3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) myopathy is seen in 6%-10% of idiopathic inflammatory myopathy and is diagnosed in the context of elevated serum creatine kinase levels, proximal muscle weakness and anti-HMGCR autoantibodies. We recently encountered a 61-year-old man with anti-HMGCR myopathy with an atypical skin manifestation, partially responsive to triple therapy with steroids, intravenous immunoglobulin (IVIG) and rituximab. To our knowledge, there have been only four reported cases of skin rash associated with anti-HMGCR myopathy. Our case demonstrates the importance of recognising atypical manifestations of anti-HMGCR myopathy. Early addition of IVIG and rituximab is also critical in patients not responding to steroid monotherapy. Delay in achieving remission leads to prolonged steroid use, lower likelihood of beginning physical therapy and overall worse clinical outcomes.


Assuntos
Doenças Musculares , Miosite , Coenzimas , Humanos , Hidroximetilglutaril-CoA Redutases , Masculino , Pessoa de Meia-Idade , Doenças Musculares/induzido quimicamente , Doenças Musculares/diagnóstico , Doenças Musculares/tratamento farmacológico , Miosite/induzido quimicamente , Miosite/diagnóstico , Miosite/tratamento farmacológico , Oxirredutases
4.
Molecules ; 26(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34361678

RESUMO

The accurate determination of analyte concentrations with selective, fast, and robust methods is the key for process control, product analysis, environmental compliance, and medical applications. Enzyme-based biosensors meet these requirements to a high degree and can be operated with simple, cost efficient, and easy to use devices. This review focuses on enzymes capable of direct electron transfer (DET) to electrodes and also the electrode materials which can enable or enhance the DET type bioelectrocatalysis. It presents amperometric biosensors for the quantification of important medical, technical, and environmental analytes and it carves out the requirements for enzymes and electrode materials in DET-based third generation biosensors. This review critically surveys enzymes and biosensors for which DET has been reported. Single- or multi-cofactor enzymes featuring copper centers, hemes, FAD, FMN, or PQQ as prosthetic groups as well as fusion enzymes are presented. Nanomaterials, nanostructured electrodes, chemical surface modifications, and protein immobilization strategies are reviewed for their ability to support direct electrochemistry of enzymes. The combination of both biosensor elements-enzymes and electrodes-is evaluated by comparison of substrate specificity, current density, sensitivity, and the range of detection.


Assuntos
Técnicas Biossensoriais/métodos , Eletrodos , Elétrons , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Biocatálise , Monitoramento Biológico/métodos , Biomarcadores Tumorais/análise , Glicemia/análise , Automonitorização da Glicemia/métodos , Coenzimas/metabolismo , Eletroquímica/métodos , Transporte de Elétrons , Estrutura Molecular , Nanoestruturas/química
5.
J Biol Chem ; 297(4): 101137, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34461093

RESUMO

In most organisms, transition metal ions are necessary cofactors of ribonucleotide reductase (RNR), the enzyme responsible for biosynthesis of the 2'-deoxynucleotide building blocks of DNA. The metal ion generates an oxidant for an active site cysteine (Cys), yielding a thiyl radical that is necessary for initiation of catalysis in all RNRs. Class I enzymes, widespread in eukaryotes and aerobic microbes, share a common requirement for dioxygen in assembly of the active Cys oxidant and a unique quaternary structure, in which the metallo- or radical-cofactor is found in a separate subunit, ß, from the catalytic α subunit. The first class I RNRs, the class Ia enzymes, discovered and characterized more than 30 years ago, were found to use a diiron(III)-tyrosyl-radical Cys oxidant. Although class Ia RNRs have historically served as the model for understanding enzyme mechanism and function, more recently, remarkably diverse bioinorganic and radical cofactors have been discovered in class I RNRs from pathogenic microbes. These enzymes use alternative transition metal ions, such as manganese, or posttranslationally installed tyrosyl radicals for initiation of ribonucleotide reduction. Here we summarize the recent progress in discovery and characterization of novel class I RNR radical-initiating cofactors, their mechanisms of assembly, and how they might function in the context of the active class I holoenzyme complex.


Assuntos
Coenzimas , Metais , Ribonucleotídeo Redutases , Animais , Catálise , Domínio Catalítico , Coenzimas/química , Coenzimas/classificação , Coenzimas/metabolismo , Humanos , Metais/química , Metais/metabolismo , Oxirredução , Ribonucleotídeo Redutases/química , Ribonucleotídeo Redutases/classificação , Ribonucleotídeo Redutases/metabolismo
6.
Biomolecules ; 11(7)2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34356667

RESUMO

During the last century, anthropogenic activities such as fertilization have led to an increase in pollution in many ecosystems by nitrogen compounds. Consequently, researchers aim to reduce nitrogen pollutants following different strategies. Some haloarchaea, owing to their denitrifier metabolism, have been proposed as good model organisms for the removal of not only nitrate, nitrite, and ammonium, but also (per)chlorates and bromate in brines and saline wastewater. Bacterial denitrification has been extensively described at the physiological, biochemical, and genetic levels. However, their haloarchaea counterparts remain poorly described. In previous work the model structure of nitric oxide reductase was analysed. In this study, a bioinformatic analysis of the sequences and the structural models of the nitrate, nitrite and nitrous oxide reductases has been described for the first time in the haloarchaeon model Haloferax mediterranei. The main residues involved in the catalytic mechanism and in the coordination of the metal centres have been explored to shed light on their structural characterization and classification. These results set the basis for understanding the molecular mechanism for haloarchaeal denitrification, necessary for the use and optimization of these microorganisms in bioremediation of saline environments among other potential applications including bioremediation of industrial waters.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Enzimas/metabolismo , Haloferax mediterranei/metabolismo , Coenzimas/metabolismo , Simulação por Computador , Desnitrificação , Enzimas/química , Haloferax mediterranei/enzimologia , Modelos Moleculares , Nitrato Redutase/química , Nitrato Redutase/metabolismo , Nitrito Redutases/química , Nitrito Redutases/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Sinais Direcionadores de Proteínas , Alinhamento de Sequência
7.
Nucleic Acids Res ; 49(15): 8822-8835, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34352100

RESUMO

The catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) contains two active sites that catalyze nucleotidyl-monophosphate transfer (NMPylation). Mechanistic studies and drug discovery have focused on RNA synthesis by the highly conserved RdRp. The second active site, which resides in a Nidovirus RdRp-Associated Nucleotidyl transferase (NiRAN) domain, is poorly characterized, but both catalytic reactions are essential for viral replication. One study showed that NiRAN transfers NMP to the first residue of RNA-binding protein nsp9; another reported a structure of nsp9 containing two additional N-terminal residues bound to the NiRAN active site but observed NMP transfer to RNA instead. We show that SARS-CoV-2 RdRp NMPylates the native but not the extended nsp9. Substitutions of the invariant NiRAN residues abolish NMPylation, whereas substitution of a catalytic RdRp Asp residue does not. NMPylation can utilize diverse nucleotide triphosphates, including remdesivir triphosphate, is reversible in the presence of pyrophosphate, and is inhibited by nucleotide analogs and bisphosphonates, suggesting a path for rational design of NiRAN inhibitors. We reconcile these and existing findings using a new model in which nsp9 remodels both active sites to alternately support initiation of RNA synthesis by RdRp or subsequent capping of the product RNA by the NiRAN domain.


Assuntos
Nidovirales/enzimologia , Nucleotídeos/metabolismo , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Coenzimas/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Difosfatos/farmacologia , Difosfonatos/farmacologia , Guanosina Trifosfato/metabolismo , Manganês , Modelos Moleculares , Nidovirales/química , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Uridina Trifosfato/metabolismo
8.
Nat Commun ; 12(1): 4742, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362891

RESUMO

The ancestors of cyanobacteria generated Earth's first biogenic molecular oxygen, but how they dealt with oxidative stress remains unconstrained. Here we investigate when superoxide dismutase enzymes (SODs) capable of removing superoxide free radicals evolved and estimate when Cyanobacteria originated. Our Bayesian molecular clocks, calibrated with microfossils, predict that stem Cyanobacteria arose 3300-3600 million years ago. Shortly afterwards, we find phylogenetic evidence that ancestral cyanobacteria used SODs with copper and zinc cofactors (CuZnSOD) during the Archaean. By the Paleoproterozoic, they became genetically capable of using iron, nickel, and manganese as cofactors (FeSOD, NiSOD, and MnSOD respectively). The evolution of NiSOD is particularly intriguing because it corresponds with cyanobacteria's invasion of the open ocean. Our analyses of metalloenzymes dealing with reactive oxygen species (ROS) now demonstrate that marine geochemical records alone may not predict patterns of metal usage by phototrophs from freshwater and terrestrial habitats.


Assuntos
Antioxidantes/metabolismo , Cianobactérias/enzimologia , Cianobactérias/metabolismo , Evolução Molecular , Teorema de Bayes , Coenzimas , Cobre , Cianobactérias/genética , Água Doce , Ferro , Manganês , Níquel/química , Estresse Oxidativo , Filogenia , Espécies Reativas de Oxigênio , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxidos , Zinco
9.
Biochem Biophys Res Commun ; 570: 15-20, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34271431

RESUMO

Glutamate dehydrogenase 3 from Candida albicans (CaGdh3) catalyzes the reversible oxidative deamination of l-glutamate, playing an important role in the yeast-to-hyphal transition of C. albicans. Here we report the crystal structures of CaGdh3 and its complex with α-ketoglutarate and NADPH. CaGdh3 exists as a hexamer, with each subunit containing two domains. The substrate and coenzyme bind in the cleft between the two domains and their binding induces a conformational change in CaGdh3. Our results will help to understand the catalytic mechanism of CaGdh3 and will provide a structural basis for the design of antifungal drugs targeting the CaGdh3 pathway.


Assuntos
Candida albicans/enzimologia , Proteínas Fúngicas/química , Glutamato Desidrogenase/química , Domínio Catalítico , Coenzimas/metabolismo , Cristalografia por Raios X , Modelos Moleculares , NADP/química , NADP/metabolismo , Conformação Proteica , Multimerização Proteica , Soluções , Especificidade por Substrato
10.
Luminescence ; 36(7): 1733-1742, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34213071

RESUMO

Glycine betaine is the main osmolyte synthesized and accumulated in mammalian renal cells. Glycine betaine synthesis is catalyzed by the enzyme betaine aldehyde dehydrogenase (BADH) using NAD+ as the coenzyme. Previous studies have shown that porcine kidney betaine aldehyde dehydrogenase (pkBADH) binds NAD+ with different affinities at each active site and that the binding is K+ dependent. The objective of this work was to analyze the changes in the pkBADH secondary and tertiary structure resulting from variable concentrations of NAD+ and the role played by K+ . Intrinsic fluorescence studies were carried out at fixed-variable concentrations of K+ and titrating the enzyme with varying concentrations of NAD+ . Fluorescence analysis showed a shift of the maximum emission towards red as the concentration of K+ was increased. Changes in the exposure of tryptophan located near the NAD+ binding site were found when the enzyme was titrated with NAD+ in the presence of potassium. Fluorescence data analysis showed that the K+ presence promoted static quenching that facilitated the pkBADH-NAD+ complex formation. DC data analysis showed that binding of K+ to the enzyme caused changes in the α-helix content of 4% and 12% in the presence of 25 mM and 100 mM K+ , respectively. The presence of K+ during NAD+ binding to pkBADH increased the thermal stability of the complex. These results indicated that K+ facilitated the pkBADH-NAD+ complex formation and suggested that K+ caused small changes in secondary and tertiary structures that could influence the active site conformation.


Assuntos
Betaína-Aldeído Desidrogenase , Potássio , Animais , Betaína-Aldeído Desidrogenase/metabolismo , Sítios de Ligação , Coenzimas , Cinética , Conformação Molecular , Suínos
11.
World J Microbiol Biotechnol ; 37(8): 130, 2021 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-34236514

RESUMO

The compound γ-aminobutyric acid (GABA) was widely used in various fields. To enhance the production of GABA in Escherichia coli BL21(DE3), the enzymes of the regeneration pathway of the coenzyme factor pyridoxal 5'-phosphate (PLP) were engineered. The recombinant E. coli strain was screened and identified. The initial concentrations of L-monosodium glutamate (L-MSG) had an obvious influence on the production of GABA. The highest concentration of GABA in recombinant E. coli BL21/pET28a-gadA was 5.54 g/L when the initial L-MSG concentration was 10 g/L, whereas it was 8.45 g/L in recombinant E. coli BL21/pET28a-gadA-SNO1-SNZ1 at an initial L-MSG concentration of 15 g/L. The corresponding conversion yields of GABA in these two strains were 91.0% and 92.7%, respectively. When the initial concentrations of L-MSG were more than 15 g/L, the concentrations of GABA in E. coli BL21/pET28a-gadA-SNO1-SNZ1 were significantly higher as compared to those in recombinant E. coli BL21/pET28a-gadA, and it reached a maximum of 13.20 g/L at an initial L-MSG concentration of 25 g/L, demonstrating that the introduction of the enzymes of the regeneration pathway of PLP favored to enhance the production of GABA. This study provides new insight into producing GABA effectively in E. coli BL21(DE3).


Assuntos
Coenzimas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfato de Piridoxal/metabolismo , Ácido gama-Aminobutírico/biossíntese , Vias Biossintéticas , Engenharia Metabólica , Glutamato de Sódio/metabolismo
12.
Chem Biol Interact ; 346: 109577, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34274336

RESUMO

The introduction of enzyme nanoreactors in medicine is relatively new. However, this technology has already been experimentally successful in cancer treatments, struggle against toxicity of reactive oxygen species in inflammatory processes, detoxification of drugs and xenobiotics, and correction of metabolic and genetic defects by using encapsulated enzymes, acting in single or cascade reactions. Biomolecules, e.g. enzymes, antibodies, reactive proteins capable of inactivating toxicants in the body are called bioscavengers. In this review, we focus on enzyme-containing nanoreactors for in vivo detoxification of organophosphorous compounds (OP) to be used for prophylaxis and post-exposure treatment of OP poisoning. A particular attention is devoted to bioscavenger-containing injectable nanoreactors operating in the bloodstream. The nanoreactor concept implements single or multiple enzymes and cofactors co-encapsulated in polymeric semi-permeable nanocontainers. Thus, the detoxification processes take place in a confined space containing highly concentrated bioscavengers. The article deals with historical and theoretical backgrounds about enzymatic detoxification of OPs in nanoreactors, nanoreactor polymeric enveloppes, realizations and advantages over other approaches using bioscavengers.


Assuntos
Nanoestruturas/química , Compostos Organofosforados/metabolismo , Xenobióticos/metabolismo , Biocatálise , Coenzimas/química , Coenzimas/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Inativação Metabólica , Nanotecnologia , Compostos Organofosforados/química , Polímeros/química
13.
Nat Chem ; 13(8): 758-765, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34183818

RESUMO

The molybdenum cofactor (Moco) is found in the active site of numerous important enzymes that are critical to biological processes. The bidentate ligand that chelates molybdenum in Moco is the pyranopterin dithiolene (molybdopterin, MPT). However, neither the mechanism of molybdate insertion into MPT nor the structure of Moco prior to its insertion into pyranopterin molybdenum enzymes is known. Here, we report this final maturation step, where adenylated MPT (MPT-AMP) and molybdate are the substrates. X-ray crystallography of the Arabidopsis thaliana Mo-insertase variant Cnx1E S269D D274S identified adenylated Moco (Moco-AMP) as an unexpected intermediate in this reaction sequence. X-ray absorption spectroscopy revealed the first coordination sphere geometry of Moco trapped in the Cnx1E active site. We have used this structural information to deduce a mechanism for molybdate insertion into MPT-AMP. Given their high degree of structural and sequence similarity, we suggest that this mechanism is employed by all eukaryotic Mo-insertases.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Proteínas de Arabidopsis/química , Coenzimas/química , Metaloproteínas/química , Molibdênio/química , Oxirredutases/química , Pteridinas/química , Arabidopsis/enzimologia , Cristalografia por Raios X , Modelos Químicos
14.
J Bacteriol ; 203(17): e0011721, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34124941

RESUMO

Archaeal methanogens, methanotrophs, and alkanotrophs have a high demand for iron (Fe) and sulfur (S); however, little is known of how they acquire, traffic, deploy, and store these elements. Here, we examined the distribution of homologs of proteins mediating key steps in Fe/S metabolism in model microorganisms, including iron(II) sensing/uptake (FeoAB), sulfide extraction from cysteine (SufS), and the biosynthesis of iron-sulfur [Fe-S] clusters (SufBCDE), siroheme (Pch2 dehydrogenase), protoheme (AhbABCD), cytochrome c (Cyt c) (CcmCF), and iron storage/detoxification (Bfr, FtrA, and IssA), among 326 publicly available, complete or metagenome-assembled genomes of archaeal methanogens/methanotrophs/alkanotrophs. The results indicate several prevalent but nonuniversal features, including FeoB, SufBC, and the biosynthetic apparatus for the basic tetrapyrrole scaffold, as well as its siroheme (and F430) derivatives. However, several early-diverging genomes lacked SufS and pathways to synthesize and deploy heme. Genomes encoding complete versus incomplete heme biosynthetic pathways exhibited equivalent prevalences of [Fe-S] cluster binding proteins, suggesting an expansion of catalytic capabilities rather than substitution of heme for [Fe-S] in the former group. Several strains with heme binding proteins lacked heme biosynthesis capabilities, while other strains with siroheme biosynthesis capability lacked homologs of known siroheme binding proteins, indicating heme auxotrophy and unknown siroheme biochemistry, respectively. While ferritin proteins involved in ferric oxide storage were widespread, those involved in storing Fe as thioferrate were unevenly distributed. Collectively, the results suggest that differences in the mechanisms of Fe and S acquisition, deployment, and storage have accompanied the diversification of methanogens/methanotrophs/alkanotrophs, possibly in response to differential availability of these elements as these organisms evolved. IMPORTANCE Archaeal methanogens, methanotrophs, and alkanotrophs, argued to be among the most ancient forms of life, have a high demand for iron (Fe) and sulfur (S) for cofactor biosynthesis, among other uses. Here, using comparative bioinformatic approaches applied to 326 genomes, we show that major differences in Fe/S acquisition, trafficking, deployment, and storage exist in this group. Variation in these characters was generally congruent with the phylogenetic placement of these genomes, indicating that variation in Fe/S usage and deployment has contributed to the diversification and ecology of these organisms. However, incongruency was observed among the distribution of cofactor biosynthesis pathways and known protein destinations for those cofactors, suggesting auxotrophy or yet-to-be-discovered pathways for cofactor biosynthesis.


Assuntos
Alcanos/metabolismo , Archaea/classificação , Archaea/metabolismo , Coenzimas/metabolismo , Ferro/metabolismo , Metano/metabolismo , Enxofre/metabolismo , Archaea/genética , Archaea/isolamento & purificação , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Processos Autotróficos , Vias Biossintéticas , Cisteína/metabolismo , Compostos Férricos/metabolismo , Heme/análogos & derivados , Heme/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Filogenia
15.
Nat Commun ; 12(1): 3436, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103525

RESUMO

Clostridioides difficile infections are an urgent medical problem. The newly discovered C. difficile adenine methyltransferase A (CamA) is specified by all C. difficile genomes sequenced to date (>300), but is rare among other bacteria. CamA is an orphan methyltransferase, unassociated with a restriction endonuclease. CamA-mediated methylation at CAAAAA is required for normal sporulation, biofilm formation, and intestinal colonization by C. difficile. We characterized CamA kinetic parameters, and determined its structure bound to DNA containing the recognition sequence. CamA contains an N-terminal domain for catalyzing methyl transfer, and a C-terminal DNA recognition domain. Major and minor groove DNA contacts in the recognition site involve base-specific hydrogen bonds, van der Waals contacts and the Watson-Crick pairing of a rearranged A:T base pair. These provide sufficient sequence discrimination to ensure high specificity. Finally, the surprisingly weak binding of the methyl donor S-adenosyl-L-methionine (SAM) might provide avenues for inhibiting CamA activity using SAM analogs.


Assuntos
Adenina/metabolismo , Clostridioides/enzimologia , DNA Bacteriano/química , Conformação de Ácido Nucleico , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Pareamento de Bases , Sequência de Bases , Coenzimas/metabolismo , Modelos Moleculares , Motivos de Nucleotídeos , S-Adenosil-Homocisteína/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , Especificidade da Espécie , Especificidade por Substrato
16.
Science ; 373(6551): 236-241, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34083449

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal agent of COVID-19, uses an RNA-dependent RNA polymerase (RdRp) for the replication of its genome and the transcription of its genes. We found that the catalytic subunit of the RdRp, nsp12, ligates two iron-sulfur metal cofactors in sites that were modeled as zinc centers in the available cryo-electron microscopy structures of the RdRp complex. These metal binding sites are essential for replication and for interaction with the viral helicase. Oxidation of the clusters by the stable nitroxide TEMPOL caused their disassembly, potently inhibited the RdRp, and blocked SARS-CoV-2 replication in cell culture. These iron-sulfur clusters thus serve as cofactors for the SARS-CoV-2 RdRp and are targets for therapy of COVID-19.


Assuntos
Coenzimas/metabolismo , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , RNA-Polimerase RNA-Dependente de Coronavírus/química , Óxidos N-Cíclicos/farmacologia , Ferro/metabolismo , SARS-CoV-2/efeitos dos fármacos , Enxofre/metabolismo , Motivos de Aminoácidos , Animais , Antivirais/farmacologia , Sítios de Ligação , Domínio Catalítico , Chlorocebus aethiops , Coenzimas/química , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Inibidores Enzimáticos/farmacologia , Ferro/química , Domínios Proteicos , RNA Helicases/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/fisiologia , Marcadores de Spin , Enxofre/química , Células Vero , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos , Zinco/metabolismo
17.
Anal Chem ; 93(27): 9602-9608, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34185503

RESUMO

Trypsin is a key proteolytic enzyme in the digestive system and its abnormal levels are indicative of some pancreatic diseases. Taking advantage of the coenzyme-mediated electrografting of ferrocenyl polymers as a novel strategy for signal amplification, herein, a signal-on cleavage-based electrochemical biosensor is reported for the highly selective interrogation of trypsin activity at ultralow levels. The construction of the trypsin biosensor involves (i) the immobilization of peptide substrates (without free carboxyl groups) via the N-terminus, (ii) the tryptic cleavage of peptide substrates, (iii) the site-specific labeling of the reversible addition-fragmentation chain transfer (RAFT) agents, and (iv) the grafting of ferrocenyl polymers through the electro-RAFT (eRAFT) polymerization, which is mediated by potentiostatic reduction of nicotinamide adenine dinucleotide (NAD+) coenzymes. Through the NAD+-mediated eRAFT (NAD+-eRAFT) polymerization of ferrocenylmethyl methacrylate (FcMMA), the presence of a few tryptic cleavage events can eventually result in the recruitment of a considerable amount of ferrocene redox tags. Obviously, the NAD+-eRAFT polymerization is low-cost and easy to operate as a highly efficient strategy for signal amplification. As expected, the as-constructed biosensor is highly selective and sensitive toward the signal-on interrogation of trypsin activity. Under optimal conditions, the detection limit can be as low as 18.2 µU/mL (∼72.8 pg/mL). The results also demonstrate that the as-constructed electrochemical trypsin biosensor is applicable to inhibitor screening and the interrogation of enzyme activity in the presence of complex sample matrices. Moreover, it is low-cost, less susceptible to false-positive results, and relatively easy to fabricate, thus holding great potential in diagnostic and therapeutic applications.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Coenzimas , Polimerização , Tripsina
18.
Chem Soc Rev ; 50(10): 5952-5984, 2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34027955

RESUMO

Racemases and epimerases catalyse changes in the stereochemical configurations of chiral centres and are of interest as model enzymes and as biotechnological tools. They also occupy pivotal positions within metabolic pathways and, hence, many of them are important drug targets. This review summarises the catalytic mechanisms of PLP-dependent, enolase family and cofactor-independent racemases and epimerases operating by a deprotonation/reprotonation (1,1-proton transfer) mechanism and methods for measuring their catalytic activity. Strategies for inhibiting these enzymes are reviewed, as are specific examples of inhibitors. Rational design of inhibitors based on substrates has been extensively explored but there is considerable scope for development of transition-state mimics and covalent inhibitors and for the identification of inhibitors by high-throughput, fragment and virtual screening approaches. The increasing availability of enzyme structures obtained using X-ray crystallography will facilitate development of inhibitors by rational design and fragment screening, whilst protein models will facilitate development of transition-state mimics.


Assuntos
Inibidores Enzimáticos/metabolismo , Racemases e Epimerases/metabolismo , Regulação Alostérica , Biocatálise , Domínio Catalítico , Coenzimas/metabolismo , Desenho de Fármacos , Inibidores Enzimáticos/química , Simulação de Dinâmica Molecular , Prótons , Racemases e Epimerases/antagonistas & inibidores , Especificidade por Substrato
19.
Arch Biochem Biophys ; 708: 108898, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-33957092

RESUMO

NAD+-linked isocitrate dehydrogenases (NAD-IDHs) catalyze the oxidative decarboxylation of isocitrate into α-ketoglutarate. Previously, we identified a novel phylogenetic clade including NAD-IDHs from several algae in the type II subfamily, represented by homodimeric NAD-IDH from Ostreococcus tauri (OtIDH). However, due to its lack of a crystalline structure, the molecular mechanisms of the ligand binding and catalysis of OtIDH are little known. Here, we elucidate four high-resolution crystal structures of OtIDH in a ligand-free and various ligand-bound forms that capture at least three states in the catalytic cycle: open, semi-closed, and fully closed. Our results indicate that OtIDH shows several novel interactions with NAD+, unlike type I NAD-IDHs, as well as a strictly conserved substrate binding mode that is similar to other homologs. The central roles of Lys283' in dual coenzyme recognition and Lys234 in catalysis were also revealed. In addition, the crystal structures obtained here also allow us to understand the catalytic mechanism. As expected, structural comparisons reveal that OtIDH has a very high structural similarity to eukaryotic NADP+-linked IDHs (NADP-IDHs) within the type II subfamily rather than with the previously reported NAD-IDHs within the type I subfamily. It has also been demonstrated that OtIDH exhibits substantial conformation changes upon ligand binding, similar to eukaryotic NADP-IDHs. These results unambiguously support our hypothesis that OtIDH and OtIDH-like homologs are possible evolutionary ancestors of eukaryotic NADP-IDHs in type II subfamily.


Assuntos
Clorófitas/enzimologia , Evolução Molecular , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/metabolismo , NADP/metabolismo , NAD/metabolismo , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Coenzimas/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Filogenia , Multimerização Proteica , Estrutura Quaternária de Proteína
20.
Appl Microbiol Biotechnol ; 105(9): 3625-3634, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33929595

RESUMO

L-tert-leucine (L-Tle) is widely used as vital chiral intermediate for pharmaceuticals and as chiral auxiliarie for organocatalysis. L-Tle is generally prepared via the asymmetric reduction of trimethylpyruvate (TMP) catalyzed by NAD+-dependent leucine dehydrogenase (LeuDH). To improve the catalytic efficiency and coenzyme affinity of LeuDH from Bacillus cereus, mutation libraries constructed by error-prone PCR and iterative saturation mutation were screened by two kinds of high-throughput methods. Compared with the wild type, the affinity of the selected mutant E24V/E116V for TMP and NADH increased by 7.7- and 2.8-fold, respectively. And the kcat/Km of E24V/E116V on TMP was 5.4-fold higher than that of the wild type. A coupled reaction comprising LeuDH with glucose dehydrogenase of Bacillus amyloliquefaciens resulted in substrate inhibition at high TMP concentrations (0.5 M), which was overcome by batch-feeding of the TMP substrate. The total turnover number and specific space-time conversion of 0.57 M substrate increased to 11,400 and 22.8 mmol·h-1·L-1·g-1, respectively. KEY POINTS: • The constructed new high-throughput screening strategy takes into account the two indicators of catalytic efficiency and coenzyme affinity. • A more efficient leucine dehydrogenase (LeuDH) mutant (E24V/E116V) was identified. • E24V/E116V has potential for the industrial synthesis of L-tert-leucine.


Assuntos
Coenzimas , Valina , Catálise , Coenzimas/metabolismo , Cinética , Leucina , Leucina Desidrogenase/genética , Leucina Desidrogenase/metabolismo , Valina/análogos & derivados
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