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1.
Biochim Biophys Acta Bioenerg ; 1859(2): 69-77, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28842179

RESUMO

The role of accessory Fe-S clusters of the F-domain in the catalytic activity of M3-type [FeFe] hydrogenase and the contribution of each of the two Fe-S surface clusters in the intermolecular electron transfer from ferredoxin are both poorly understood. We designed, constructed, produced and spectroscopically, electrochemically and biochemically characterized three mutants of Clostridium acetobutylicum CaHydA hydrogenase with modified Fe-S clusters: two site-directed mutants, HydA_C100A and HydA_C48A missing the FS4C and the FS2 surface Fe-S clusters, respectively, and a HydA_ΔDA mutant that completely lacks the F-domain. Analysis of the mutant enzyme activities clearly demonstrated the importance of accessory clusters in retaining full enzyme activity at potentials around and higher than the equilibrium 2H+/H2 potential but not at the lowest potentials, where all enzymes have a similar turnover rate. Moreover, our results, combined with molecular modelling approaches, indicated that the FS2 cluster is the main gate for electron transfer from reduced ferredoxin.


Assuntos
Clostridium acetobutylicum/enzimologia , Hidrogenase/química , Substituição de Aminoácidos , Proteínas de Bactérias , Clostridium acetobutylicum/genética , Hidrogenase/genética , Mutação de Sentido Incorreto , Domínios Proteicos
3.
J Am Chem Soc ; 140(16): 5516-5526, 2018 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-29595965

RESUMO

[FeFe]-hydrogenases, HydAs, are unique biocatalysts for proton reduction to H2. However, they suffer from a number of drawbacks for biotechnological applications: size, number and diversity of metal cofactors, oxygen sensitivity. Here we show that HydA from Megasphaera elsdenii (MeHydA) displays significant resistance to O2. Furthermore, we produced a shorter version of this enzyme (MeH-HydA), lacking the N-terminal domain harboring the accessory FeS clusters. As shown by detailed spectroscopic and biochemical characterization, MeH-HydA displays the following interesting properties. First, a functional active site can be assembled in MeH-HydA in vitro, providing the enzyme with excellent hydrogenase activity. Second, the resistance of MeHydA to O2 is conserved in MeH-HydA. Third, MeH-HydA is more biased toward proton reduction than MeHydA, as the result of the truncation changing the rate limiting steps in catalysis. This work shows that it is possible to engineer HydA to generate an active hydrogenase that combines the resistance of the most resistant HydAs and the simplicity of algal HydAs, containing only the H-cluster.


Assuntos
Hidrogenase/metabolismo , Megasphaera elsdenii/enzimologia , Oxigênio/metabolismo , Engenharia de Proteínas , Biocatálise , Monóxido de Carbono/metabolismo , Domínio Catalítico , Hidrogenase/química , Hidrogenase/genética , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Megasphaera elsdenii/química , Megasphaera elsdenii/genética , Megasphaera elsdenii/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Engenharia de Proteínas/métodos
4.
Metab Eng ; 40: 138-147, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28159643

RESUMO

Clostridium acetobutylicum possesses two homologous buk genes, buk (or buk1) and buk2, which encode butyrate kinases involved in the last step of butyrate formation. To investigate the contribution of buk in detail, an in-frame deletion mutant was constructed. However, in all the Δbuk mutants obtained, partial deletions of the upstream ptb gene were observed, and low phosphotransbutyrylase and butyrate kinase activities were measured. This demonstrates that i) buk (CA_C3075) is the key butyrate kinase-encoding gene and that buk2 (CA_C1660) that is poorly transcribed only plays a minor role; and ii) strongly suggests that a Δbuk mutant is not viable if the ptb gene is not also inactivated, probably due to the accumulation of butyryl-phosphate, which might be toxic for the cell. One of the ΔbukΔptb mutants was subjected to quantitative transcriptomic (mRNA molecules/cell) and fluxomic analyses in acidogenic, solventogenic and alcohologenic chemostat cultures. In addition to the low butyrate production, drastic changes in metabolic fluxes were also observed for the mutant: i) under acidogenic conditions, the primary metabolite was butanol and a new metabolite, 2-hydroxy-valerate, was produced ii) under solventogenesis, 58% increased butanol production was obtained compared to the control strain under the same conditions, and a very high yield of butanol formation (0.3gg-1) was reached; and iii) under alcohologenesis, the major product was lactate. Furthermore, at the transcriptional level, adhE2, which encodes an aldehyde/alcohol dehydrogenase and is known to be a gene specifically expressed in alcohologenesis, was surprisingly highly expressed in all metabolic states in the mutant. The results presented here not only support the key roles of buk and ptb in butyrate formation but also highlight the metabolic flexibility of C. acetobutylicum in response to genetic alteration of its primary metabolism.


Assuntos
Ácido Butírico/metabolismo , Clostridium acetobutylicum/fisiologia , Redes e Vias Metabólicas/fisiologia , Fosfato Acetiltransferase/metabolismo , Fosfotransferases (Aceptor do Grupo Carboxila)/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Engenharia Metabólica/métodos , Análise do Fluxo Metabólico/métodos , Mutação/genética , Fosfato Acetiltransferase/genética , Fosfotransferases (Aceptor do Grupo Carboxila)/genética
5.
J Am Chem Soc ; 138(41): 13612-13618, 2016 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-27649394

RESUMO

FeFe hydrogenases catalyze H2 oxidation and formation at an inorganic active site (the "H-cluster"), which consists of a [Fe2(CO)3(CN)2(dithiomethylamine)] subcluster covalently attached to a Fe4S4 subcluster. This active site is photosensitive: visible light has been shown to induce the release of exogenous CO (a reversible inhibitor of the enzyme), shuffle the intrinsic CO ligands, and even destroy the H-cluster. These reactions must be understood because they may negatively impact the use of hydrogenase for the photoproduction of H2. Here, we explore in great detail the reactivity of the excited states of the H-cluster under catalytic conditions by examining, both experimentally and using TDDFT calculations, the simplest photochemical reaction: the binding and release of exogenous CO. A simple dyad model can be used to predict which excitations are active. This strategy could be used for probing other aspects of the photoreactivity of the H-cluster.

6.
J Am Chem Soc ; 137(39): 12580-7, 2015 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-26352172

RESUMO

The mechanism of reaction of FeFe hydrogenases with oxygen has been debated. It is complex, apparently very dependent on the details of the protein structure, and difficult to study using conventional kinetic techniques. Here we build on our recent work on the anaerobic inactivation of the enzyme [Fourmond et al. Nat. Chem. 2014, 4, 336-342] to propose and apply a new method for studying this reaction. Using electrochemical measurements of the turnover rate of hydrogenase, we could resolve the first steps of the inhibition reaction and accurately determine their rates. We show that the two most studied FeFe hydrogenases, from Chlamydomonas reinhardtii and Clostridium acetobutylicum, react with O2 according to the same mechanism, despite the fact that the former is much more O2 sensitive than the latter. Unlike often assumed, both enzymes are reversibly inhibited by a short exposure to O2. This will have to be considered to elucidate the mechanism of inhibition, before any prediction can be made regarding which mutations will improve oxygen resistance. We hope that the approach described herein will prove useful in this respect.


Assuntos
Hidrogenase/antagonistas & inibidores , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/antagonistas & inibidores , Proteínas Ferro-Enxofre/metabolismo , Modelos Moleculares , Oxigênio/química , Domínio Catalítico , Eletroquímica , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Cinética
7.
Blood ; 120(17): 3390-2, 2012 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-23100301

RESUMO

Two articles in this issue of Blood from Feys et al and Callewaert et al, respectively, have employed very similar and elegant strategies in attempts to ameliorate the symptoms of thrombotic thrombocytopenic purpura (TTP).


Assuntos
Anemia Hemolítica/tratamento farmacológico , Anticorpos Monoclonais/uso terapêutico , Fibrinolíticos/uso terapêutico , Complexo Glicoproteico GPIb-IX de Plaquetas/antagonistas & inibidores , Púrpura Trombocitopênica Trombótica/tratamento farmacológico , Fator de von Willebrand/antagonistas & inibidores , Animais
8.
Xenotransplantation ; 21(3): 274-86, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24628649

RESUMO

BACKGROUND: Although transplantation of genetically modified porcine livers into baboons has yielded recipient survival for up to 7 days, survival is limited by profound thrombocytopenia, which becomes manifest almost immediately after revascularization, and by subsequent coagulopathy. Porcine von Willebrand's factor (VWF), a glycoprotein that adheres to activated platelets to initiate thrombus formation, has been shown to constitutively activate human platelets via their glycoprotein Ib (GPIb) receptors. Here, we report our pig-to-primate liver xenoperfusion model and evaluate whether targeting the GPIb-VWF axis prevents platelet sequestration. METHODS: Twelve baboons underwent cross-circulation with the following extracorporeal livers: one allogeneic control with a baboon liver, 4 xenogeneic controls with a GalTKO.hCD46 pig liver, 3 GalTKO.hCD46 pig livers in recipients treated with αGPIb antibody during perfusion, and 4 GalTKO.hCD46 pig livers pre-treated with D-arginine vasopressin (DDAVP) in recipients treated with αGPIb antibody during perfusion. RESULTS: All perfused livers appeared grossly and macroscopically normal and produced bile. Xenograft liver perfusion experiments treated with αGPIb antibody may show less platelet sequestration during the initial 2 h of perfusion. Portal venous resistance remained constant in all perfusion experiments. Platelet activation studies demonstrated platelet activation in all xenoperfusions, but not in the allogeneic perfusion. CONCLUSION: These observations suggest that primate platelet sequestration by porcine liver and the associated thrombocytopenia are multifactorial and perhaps partially mediated by a constitutive interaction between porcine VWF and the primate GPIb receptor. Control of platelet sequestration and consumptive coagulopathy in liver xenotransplantation will likely require a multifaceted approach in our clinically relevant perfusion model.


Assuntos
Fragmentos Fab das Imunoglobulinas/uso terapêutico , Imunossupressores/uso terapêutico , Transplante de Fígado/métodos , Complexo Glicoproteico GPIb-IX de Plaquetas/imunologia , Complicações Pós-Operatórias/prevenção & controle , Trombocitopenia/prevenção & controle , Transplante Heterólogo/métodos , Animais , Animais Geneticamente Modificados , Biomarcadores/metabolismo , Circulação Extracorpórea , Galactosiltransferases/genética , Técnicas de Inativação de Genes , Sobrevivência de Enxerto , Humanos , Proteína Cofatora de Membrana/genética , Papio , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Complicações Pós-Operatórias/etiologia , Suínos/genética , Trombocitopenia/etiologia , Fator de von Willebrand/metabolismo
9.
Proc Natl Acad Sci U S A ; 108(4): 1278-83, 2011 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-21205901

RESUMO

Bacterial metabolism is characterized by a remarkable capacity to rapidly adapt to environmental changes. We restructured the central metabolic network in Escherichia coli to force a higher production of NADPH, and then grew this strain in conditions favoring adaptive evolution. A six-fold increase in growth capacity was attained that could be attributed in multiple clones, after whole genome mutation mapping, to a specific single mutation. Each clone had an evolved NuoF*(E183A) enzyme in the respiratory complex I that can now oxidize both NADH and NADPH. When a further strain was constructed with an even higher degree of NADPH stress such that growth was impossible on glucose mineral medium, a solid-state screening for mutations restoring growth, led to two different types of NuoF mutations in strains having recovered growth capacity. In addition to the previously seen E183A mutation other clones showed a E183G mutation, both having NADH and NADPH oxidizing ability. These results demonstrate the unique solution used by E. coli to overcome the NADPH stress problem. This solution creates a new function for NADPH that is no longer restricted to anabolic synthesis reactions but can now be also used to directly produce catabolic energy.


Assuntos
Adaptação Fisiológica/genética , Proteínas de Escherichia coli/genética , Mutação , Quinona Redutases/genética , Aerobiose , Substituição de Aminoácidos , Sítios de Ligação , Biocatálise , Evolução Molecular Direcionada , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Genoma Bacteriano/genética , Glucose/metabolismo , Cinética , Modelos Moleculares , NAD/metabolismo , NADP/metabolismo , Oxirredução , Fenótipo , Estrutura Terciária de Proteína , Quinona Redutases/química , Quinona Redutases/metabolismo , Estresse Fisiológico
10.
J Am Chem Soc ; 135(10): 3926-38, 2013 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-23362993

RESUMO

Using direct electrochemistry to learn about the mechanism of electrocatalysts and redox enzymes requires that kinetic models be developed. Here we thoroughly discuss the interpretation of electrochemical signals obtained with adsorbed enzymes and molecular catalysts that can reversibly convert their substrate and product. We derive analytical relations between electrochemical observables (overpotentials for catalysis in each direction, positions, and magnitudes of the features of the catalytic wave) and the characteristics of the catalytic cycle (redox properties of the catalytic intermediates, kinetics of intramolecular and interfacial electron transfer, etc.). We discuss whether or not the position of the wave is determined by the redox potential of a redox relay when intramolecular electron transfer is slow. We demonstrate that there is no simple relation between the reduction potential of the active site and the catalytic bias of the enzyme, defined as the ratio of the oxidative and reductive limiting currents; this explains the recent experimental observation that the catalytic bias of NiFe hydrogenase depends on steps of the catalytic cycle that occur far from the active site [Abou Hamdan et al., J. Am. Chem. Soc. 2012, 134, 8368]. On the experimental side, we examine which models can best describe original data obtained with various NiFe and FeFe hydrogenases, and we illustrate how the presence of an intramolecular electron transfer chain affects the voltammetry by comparing the data obtained with the FeFe hydrogenases from Chlamydomonas reinhardtii and Clostridium acetobutylicum, only one of which has a chain of redox relays. The considerations herein will help the interpretation of electrochemical data previously obtained with various other bidirectional oxidoreductases, and, possibly, synthetic inorganic catalysts.


Assuntos
Técnicas Eletroquímicas , Elétrons , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Biocatálise , Chlamydomonas reinhardtii/enzimologia , Clostridium acetobutylicum/enzimologia , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Modelos Moleculares , Oxirredução
11.
Metab Eng ; 18: 1-8, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23541907

RESUMO

Clostridium acetobutylicum was metabolically engineered to produce a biofuel consisting of an isopropanol/butanol/ethanol mixture. For this purpose, different synthetic isopropanol operons were constructed and introduced on plasmids in a butyrate minus mutant strain (C. acetobutylicum ATCC 824 Δcac15ΔuppΔbuk). The best strain expressing the isopropanol operon from the thl promoter was selected from batch experiments at pH 5. By further optimizing the pH of the culture, a biofuel mixture with almost no by-products was produced at a titer, a yield and productivity never reached before, opening the opportunities to develop an industrial process for alternative biofuels with Clostridial species. Furthermore, by performing in vivo and in vitro flux analysis of the synthetic isopropanol pathway, this flux was identified to be limited by the [acetate](int) and the high Km of CoA-transferase for acetate. Decreasing the Km of this enzyme using a protein engineering approach would be a good target for improving isopropanol production and avoiding acetate accumulation in the culture medium.


Assuntos
2-Propanol/metabolismo , Biocombustíveis , Butanóis/metabolismo , Clostridium acetobutylicum , Etanol/metabolismo , Engenharia Metabólica , Clostridium acetobutylicum/genética , Clostridium acetobutylicum/crescimento & desenvolvimento , Clostridium acetobutylicum/metabolismo , Concentração de Íons de Hidrogênio , Óperon/genética , Plasmídeos/genética
12.
Anal Chem ; 84(18): 7999-8005, 2012 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-22891965

RESUMO

Direct electron transfer between enzymes and electrodes is now commonly achieved, but obtaining protein films that are very stable may be challenging. This is particularly crucial in the case of hydrogenases, the enzymes that catalyze the biological conversion between dihydrogen and protons, because the instability of the hydrogenase films may prevent the use of these enzymes as electrocatalysts of H(2) oxidation and production in biofuel cells and photoelectrochemical cells. Here we show that two different FeFe hydrogenases (from Chamydomonas reinhardtii and Clostridium acetobutylicum) can be covalently attached to functionalized pyrolytic graphite electrodes using peptidic coupling. In both cases, a surface patch of lysine residues makes it possible to favor an orientation that is efficient for fast, direct electron transfer. High hydrogen-oxidation current densities are maintained for up to one week, the only limitation being the intrinsic stability of the enzyme. We also show that covalent attachment has no effect on the catalytic properties of the enzyme, which means that this strategy can also used be for electrochemical studies of the catalytic mechanism.


Assuntos
Carbono/química , Técnicas Eletroquímicas , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Biocatálise , Fontes de Energia Bioelétrica , Chlamydomonas reinhardtii/enzimologia , Clostridium acetobutylicum/enzimologia , Eletrodos , Transporte de Elétrons , Hidrogênio/metabolismo , Hidrogenase/química , Proteínas Ferro-Enxofre/química , Oxirredução , Prótons
13.
Blood ; 116(22): 4646-56, 2010 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-20833976

RESUMO

Within the healthy population, there is substantial, heritable, and interindividual variability in the platelet response. We explored whether a proportion of this variability could be accounted for by interindividual variation in gene expression. Through a correlative analysis of genome-wide platelet RNA expression data from 37 subjects representing the normal range of platelet responsiveness within a cohort of 500 subjects, we identified 63 genes in which transcript levels correlated with variation in the platelet response to adenosine diphosphate and/or the collagen-mimetic peptide, cross-linked collagen-related peptide. Many of these encode proteins with no reported function in platelets. An association study of 6 of the 63 genes in 4235 cases and 6379 controls showed a putative association with myocardial infarction for COMMD7 (COMM domain-containing protein 7) and a major deviation from the null hypo thesis for LRRFIP1 [leucine-rich repeat (in FLII) interacting protein 1]. Morpholino-based silencing in Danio rerio identified a modest role for commd7 and a significant effect for lrrfip1 as positive regulators of thrombus formation. Proteomic analysis of human platelet LRRFIP1-interacting proteins indicated that LRRFIP1 functions as a component of the platelet cytoskeleton, where it interacts with the actin-remodeling proteins Flightless-1 and Drebrin. Taken together, these data reveal novel proteins regulating the platelet response.


Assuntos
Plaquetas/metabolismo , Perfilação da Expressão Gênica , Proteínas de Ligação a RNA/metabolismo , Animais , Inativação Gênica , Genótipo , Humanos , Ativação Plaquetária , Proteoma/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Trombose , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
14.
Nat Chem Biol ; 6(1): 63-70, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19966788

RESUMO

In hydrogenases and many other redox enzymes, the buried active site is connected to the solvent by a molecular channel whose structure may determine the enzyme's selectivity with respect to substrate and inhibitors. The role of these channels has been addressed using crystallography and molecular dynamics, but kinetic data are scarce. Using protein film voltammetry, we determined and then compared the rates of inhibition by CO and O2 in ten NiFe hydrogenase mutants and two FeFe hydrogenases. We found that the rate of inhibition by CO is a good proxy of the rate of diffusion of O2 toward the active site. Modifying amino acids whose side chains point inside the tunnel can slow this rate by orders of magnitude. We quantitatively define the relations between diffusion, the Michaelis constant for H2 and rates of inhibition, and we demonstrate that certain enzymes are slowly inactivated by O2 because access to the active site is slow.


Assuntos
Desulfovibrio/enzimologia , Hidrogenase/química , Oxigênio/química , Aminoácidos/química , Monóxido de Carbono/química , Domínio Catalítico , Cristalografia por Raios X/métodos , Difusão , Eletroquímica/métodos , Espectroscopia de Ressonância de Spin Eletrônica , Hidrogênio/química , Cinética , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular
15.
Biotechnol Adv ; 54: 107781, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34029623

RESUMO

Tetanus vaccination is of major importance for public health in most countries in the world. The World Health Organization indicated that 15,000 tetanus cases were reported in 2018 (Organization, World Health, 2019). Currently, vaccine manufacturers use tetanus toxin produced by Clostridium tetani fermentation in complex media. The complex components, commonly derived from animal sources, introduce potential variability in cultures. To achieve replicable fermentation and to avoid toxic or allergic reactions from animal-source compounds, several studies have tried to switch from complex to chemically defined media without affecting toxin titers. The present review introduces the current knowledge on i) C. tetani strain diversity, whole-genome sequences and metabolic networks; ii) toxin regulation and synthesis; and iii) culture media, cultivation processes and growth requirements. We critically reviewed the reported data on metabolism in C. tetani and completed comparative genomic and proteomic analyses with other Clostridia species. We integrated genomic data based on whole-genome sequence annotation, supplemented with cofactor specificities determined by protein sequence identity, in a new map of C. tetani central metabolism. This is the first data review that integrates insights from omics experiments on C. tetani. The overview of C. tetani physiology described here could provide support for the design of new chemically defined media devoid of complex sources for toxin production.


Assuntos
Clostridium tetani , Proteômica , Animais , Reatores Biológicos , Clostridium , Clostridium tetani/genética , Clostridium tetani/metabolismo , Toxina Tetânica/genética , Toxina Tetânica/metabolismo
16.
Nat Commun ; 13(1): 4691, 2022 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-35948538

RESUMO

Clostridium acetobutylicum is a promising biocatalyst for the renewable production of n-butanol. Several metabolic strategies have already been developed to increase butanol yields, most often based on carbon pathway redirection. However, it has previously demonstrated that the activities of both ferredoxin-NADP+ reductase and ferredoxin-NAD+ reductase, whose encoding genes remain unknown, are necessary to produce the NADPH and the extra NADH needed for butanol synthesis under solventogenic conditions. Here, we purify, identify and partially characterize the proteins responsible for both activities and demonstrate the involvement of the identified enzymes in butanol synthesis through a reverse genetic approach. We further demonstrate the yield of butanol formation is limited by the level of expression of CA_C0764, the ferredoxin-NADP+ reductase encoding gene and the bcd operon, encoding a ferredoxin-NAD+ reductase. The integration of these enzymes into metabolic engineering strategies introduces opportunities for developing a homobutanologenic C. acetobutylicum strain.


Assuntos
Clostridium acetobutylicum , Butanóis/metabolismo , Clostridium/metabolismo , Clostridium acetobutylicum/genética , Clostridium acetobutylicum/metabolismo , Elétrons , Fermentação , Ferredoxina-NADP Redutase/metabolismo , Ferredoxinas/metabolismo , NAD/metabolismo , NADP/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo
17.
J Bacteriol ; 193(12): 3127-34, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21478343

RESUMO

The glycerol oxidative pathway of Clostridium butyricum VPI 1718 plays an important role in glycerol dissimilation. We isolated, sequenced, and characterized the region coding for the glycerol oxidation pathway. Five open reading frames (ORFs) were identified: dhaR, encoding a putative transcriptional regulator; dhaD (1,142 bp), encoding a glycerol dehydrogenase; and dhaK (995 bp), dhaL (629 bp), and dhaM (386 bp), encoding a phosphoenolpyruvate (PEP)-dependent dihydroxyacetone (DHA) kinase enzyme complex. Northern blot analysis demonstrated that the last four genes are transcribed as a 3.2-kb polycistronic operon only in glycerol-metabolizing cultures, indicating that the expression of this operon is regulated at the transcriptional level. The transcriptional start site of the operon was determined by primer extension, and the promoter region was deduced. The glycerol dehydrogenase activity of DhaD and the PEP-dependent DHA kinase activity of DhaKLM were demonstrated by heterologous expression in different Escherichia coli mutants. Based on our complementation experiments, we proposed that the HPr phosphoryl carrier protein and His9 residue of the DhaM subunit are involved in the phosphoryl transfer to dihydroxyacetone-phosphate. DhaR, a potential regulator of this operon, was found to contain conserved transmitter and receiver domains that are characteristic of two-component systems present in the AraC family. To the best of our knowledge, this is the first molecular characterization of a glycerol oxidation pathway in a Gram-positive bacterium.


Assuntos
Proteínas de Bactérias/metabolismo , Clostridium butyricum/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Glicerol/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Família Multigênica , Oxirredução , Transcrição Gênica
18.
J Am Chem Soc ; 133(7): 2096-9, 2011 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-21271703

RESUMO

Carbon monoxide is often described as a competitive inhibitor of FeFe hydrogenases, and it is used for probing H(2) binding to synthetic or in silico models of the active site H-cluster. Yet it does not always behave as a simple inhibitor. Using an original approach which combines accurate electrochemical measurements and theoretical calculations, we elucidate the mechanism by which, under certain conditions, CO binding can cause permanent damage to the H-cluster. Like in the case of oxygen inhibition, the reaction with CO engages the entire H-cluster, rather than only the Fe(2) subsite.


Assuntos
Monóxido de Carbono/química , Hidrogenase/química , Teoria Quântica , Domínio Catalítico , Eletroquímica , Oxirredução
19.
Blood ; 114(24): 5044-51, 2009 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-19741191

RESUMO

Xenotransplantation systems have been used with increasing success to better understand human hematopoiesis and thrombopoiesis. In this study, we demonstrate that production of human platelets in nonobese diabetic/severe combined immunodeficient mice after transplantation of unexpanded cord-blood CD34(+) cells was detected within 10 days after transplantation, with the number of circulating human platelets peaking at 2 weeks (up to 87 x 10(3)/microL). This rapid human platelet production was followed by a second wave of platelet formation 5 weeks after transplantation, with a population of 5% still detected after 8 weeks, attesting for long-term engraftment. Platelets issued from human hematopoietic stem cell progenitors are functional, as assessed by increased CD62P expression and PAC1 binding in response to collagen-related peptide and thrombin receptor-activating peptide activation and their ability to incorporate into thrombi formed on a collagen-coated surface in an ex vivo flow model of thrombosis. This interaction was abrogated by addition of inhibitory monoclonal antibodies against human glycoprotein Ibalpha (GPIbalpha) and GPIIb/IIIa. Thus, our mouse model with production of human platelets may be further explored to study the function of genetically modified platelets, but also to investigate the effect of stimulators or inhibitors of human thrombopoiesis in vivo.


Assuntos
Plaquetas/fisiologia , Diferenciação Celular/fisiologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Modelos Animais de Doenças , Trombopoese/fisiologia , Animais , Antígenos CD34/metabolismo , Plaquetas/citologia , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Trombose/fisiopatologia
20.
Blood ; 113(19): 4754-62, 2009 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-19109564

RESUMO

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)-based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.


Assuntos
Plaquetas/metabolismo , Genômica , Proteínas de Membrana/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Trombose/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Western Blotting , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Perfilação da Expressão Gênica , Humanos , Lasers , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Agregação Plaquetária , Trombose/etiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética
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