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1.
PLoS Genet ; 15(10): e1008435, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31613892

RESUMO

Bacteria have evolved sophisticated uptake machineries in order to obtain the nutrients required for growth. Gram-negative plant pathogens of the genus Pectobacterium obtain iron from the protein ferredoxin, which is produced by their plant hosts. This iron-piracy is mediated by the ferredoxin uptake system (Fus), a gene cluster encoding proteins that transport ferredoxin into the bacterial cell and process it proteolytically. In this work we show that gene clusters related to the Fus are widespread in bacterial species. Through structural and biochemical characterisation of the distantly related Fus homologues YddB and PqqL from Escherichia coli, we show that these proteins are analogous to components of the Fus from Pectobacterium. The membrane protein YddB shares common structural features with the outer membrane ferredoxin transporter FusA, including a large extracellular substrate binding site. PqqL is an active protease with an analogous periplasmic localisation and iron-dependent expression to the ferredoxin processing protease FusC. Structural analysis demonstrates that PqqL and FusC share specific features that distinguish them from other members of the M16 protease family. Taken together, these data provide evidence that protease associated import systems analogous to the Fus are widespread in Gram-negative bacteria.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Membrana Transportadoras/genética , Pectobacterium/genética , Peptídeo Hidrolases/genética , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/genética , Ferredoxinas/metabolismo , Genes Bacterianos/fisiologia , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Família Multigênica/fisiologia , Óperon/fisiologia , Pectobacterium/metabolismo , Peptídeo Hidrolases/metabolismo
2.
Biochim Biophys Acta Bioenerg ; 1860(11): 148084, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31520614

RESUMO

Photosynthetic [2Fe-2S] plant-type ferredoxins have a central role in electron transfer between the photosynthetic chain and various metabolic pathways. Several genes are coding for [2Fe2S] ferredoxins in cyanobacteria, with four in the thermophilic cyanobacterium Thermosynechococcus elongatus. The structure and functional properties of the major ferredoxin Fd1 are well known but data on the other ferredoxins are scarce. We report the structural and functional properties of a novel minor type ferredoxin, Fd2 of T. elongatus, homologous to Fed4 from Synechocystis sp. PCC 6803. Remarkably, the midpoint potential of Fd2, Em = -440 mV, is lower than that of Fd1, Em = -372 mV. However, while Fd2 can efficiently react with photosystem I or nitrite reductase, time-resolved spectroscopy shows that Fd2 has a very low capacity to reduce ferredoxin-NADP+ oxidoreductase (FNR). These unique Fd2 properties are discussed in relation with its structure, solved at 1.38 Šresolution. The Fd2 structure significantly differs from other known ferredoxins structures in loop 2, N-terminal region, hydrogen bonding networks and surface charge distributions. UV-Vis, EPR, and Mid- and Far-IR data also show that the electronic properties of the [2Fe2S] cluster of Fd2 and its interaction with the protein differ from those of Fd1 both in the oxidized and reduced states. The structural analysis allows to propose that valine in the motif Cys53ValAsnCys56 of Fd2 and the specific orientation of Phe72, explain the electron transfer properties of Fd2. Strikingly, the nature of these residues correlates with different phylogenetic groups of cyanobacterial Fds. With its low redox potential and its discrimination against FNR, Fd2 exhibits a unique capacity to direct efficiently photosynthetic electrons to metabolic pathways not dependent on FNR.


Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , Ferredoxinas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cianobactérias/genética , Ferredoxinas/química , Ferredoxinas/genética , Ligações de Hidrogênio , Cinética , Modelos Moleculares , Filogenia , Alinhamento de Sequência
3.
Nat Commun ; 10(1): 3566, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31395877

RESUMO

Iron-sulfur (Fe-S) clusters are essential protein cofactors whose biosynthetic defects lead to severe diseases among which is Friedreich's ataxia caused by impaired expression of frataxin (FXN). Fe-S clusters are biosynthesized on the scaffold protein ISCU, with cysteine desulfurase NFS1 providing sulfur as persulfide and ferredoxin FDX2 supplying electrons, in a process stimulated by FXN but not clearly understood. Here, we report the breakdown of this process, made possible by removing a zinc ion in ISCU that hinders iron insertion and promotes non-physiological Fe-S cluster synthesis from free sulfide in vitro. By binding zinc-free ISCU, iron drives persulfide uptake from NFS1 and allows persulfide reduction into sulfide by FDX2, thereby coordinating sulfide production with its availability to generate Fe-S clusters. FXN stimulates the whole process by accelerating persulfide transfer. We propose that this reconstitution recapitulates physiological conditions which provides a model for Fe-S cluster biosynthesis, clarifies the roles of FDX2 and FXN and may help develop Friedreich's ataxia therapies.


Assuntos
Ferredoxinas/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Proteínas com Ferro-Enxofre/metabolismo , Sulfetos/metabolismo , Liases de Carbono-Enxofre/metabolismo , Ferredoxinas/isolamento & purificação , Ataxia de Friedreich/patologia , Ferro/metabolismo , Proteínas de Ligação ao Ferro/isolamento & purificação , Proteínas com Ferro-Enxofre/química , Proteínas com Ferro-Enxofre/genética , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Espectroscopia de Prótons por Ressonância Magnética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Zinco/metabolismo
4.
Biochim Biophys Acta Bioenerg ; 1860(10): 148063, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31419396

RESUMO

Ferritin-like proteins, Dps (DNA-binding protein from starved cells), store iron and play a key role in the iron homeostasis in bacteria, yet their iron releasing machinery remains largely unexplored. The electron donor proteins that may interact with Dps and promote the mobilization of the stored iron have hitherto not been identified. Here, we investigate the binding capacity of the two atypical Dps proteins NpDps4 and NpDps5 from Nostoc punctiforme to isolated ferredoxins. We report NpDps-ferredoxin interactions by fluorescence correlation spectroscopy (FCS) and fluorescence resonance energy transfer (FRET) methods. Dynamic light scattering, size exclusion chromatography and native gel electrophoresis results show that NpDps4 forms a dodecamer at both pH 6.0 and pH 8.0, while NpDps5 forms a dodecamer only at pH 6.0. In addition, FCS data clearly reveal that the non-canonical NpDps5 interacts with DNA at pH 6.0. Our spectroscopic analysis shows that [FeS] centers of the three recombinantly expressed and isolated ferredoxins are properly incorporated and are consistent with their respective native states. The results support our hypothesis that ferredoxins could be involved in cellular iron homeostasis by interacting with Dps and assisting the release of stored iron.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , DNA/metabolismo , Ferredoxinas/metabolismo , Nostoc/metabolismo , Proteínas de Bactérias/metabolismo , Cianobactérias , Proteínas de Ligação a DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Concentração de Íons de Hidrogênio , Ferro/metabolismo , Ligação Proteica , Multimerização Proteica , Espectrometria de Fluorescência
5.
Endocrinology ; 160(10): 2401-2416, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31322700

RESUMO

The roles of steroids in zebrafish sex differentiation, gonadal development, and function of the adult gonad are poorly understood. Herein, we used ferredoxin 1b (fdx1b) mutant zebrafish to explore such processes. Fdx1b is an essential electron-providing cofactor to mitochondrial steroidogenic enzymes, which are crucial for glucocorticoid and androgen production in vertebrates. Fdx1b-/- zebrafish mutants develop into viable adults in which concentrations of androgens and cortisol are significantly reduced. Adult fdx1b-/- mutant zebrafish display predominantly female secondary sex characteristics but may possess either ovaries or testes, confirming that androgen signaling is dispensable for testicular differentiation in this species, as previously demonstrated in androgen receptor mutant zebrafish. Adult male fdx1b-/- mutant zebrafish exhibit reduced characteristic breeding behaviors and impaired sperm production, resulting in infertility in standard breeding scenarios. However, eggs collected from wild-type females can be fertilized by the sperm of fdx1b-/- mutant males by in vitro fertilization. The testes of fdx1b-/- mutant males are disorganized and lack defined seminiferous tubule structure. Expression of several promale and spermatogenic genes is decreased in the testes of fdx1b-/- mutant males, including promale transcription factor sox9a and spermatogenic genes igf3 and insl3. This study establishes an androgen- and cortisol-deficient fdx1b zebrafish mutant as a model for understanding the effects of steroid deficiency on sex development and reproductive function. This model will be particularly useful for further investigation of the roles of steroids in spermatogenesis, gonadal development, and regulation of reproductive behavior, thus enabling further elucidation of the physiological consequences of endocrine disruption in vertebrates.


Assuntos
Ferredoxinas/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Testículo/anormalidades , Proteínas de Peixe-Zebra/metabolismo , Animais , Feminização/genética , Ferredoxinas/metabolismo , Infertilidade Masculina , Masculino , Diferenciação Sexual/genética , Desenvolvimento Sexual , Espermatogênese , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
6.
Biochim Biophys Acta Bioenerg ; 1860(9): 689-698, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31336103

RESUMO

The binding of FNR to PSI has been postulated long ago, however, a clear evidence is still missing. In this work, using isothermal titration calorimetry (ITC), we found that FNR binds to photosystem I with its light harvesting complex I (PSI-LHCI) from C. reinhardtii with a 1:1 stoichiometry, a Kd of ~0.8 µM and ∆H of -20.7 kcal/mol. Titrations at different temperatures were used to determine the heat capacity change, ∆CP, of the binding, through which the size of the interface area between the proteins was assessed as ~3000 Å2. In a different set of ITC experiments, introduction of various sucrose concentrations was used to estimate that ~95 water molecules are released to the solvent. These observations support the notion of a binding site shared by few of the photosystem I - light harvesting complex I (PSI-LHCI) subunits in addition to PsaE. Based on these results, a hypothetical model was built for the binding site of FNR at PSI, using known crystallographic structures of: cyanobacterial PSI in complex with ferredoxin (Fd), plant PSI-LHCI and Fd:FNR complex from cyanobacteria. FNR binding site location is proposed to be at the foot of the stromal ridge and above the inner LHCI belt. It is expected to form contacts with PsaE, PsaB, PsaF and at least one of the LHCI. In addition, a ~4.5-fold increased affinity between FNR and PSI-LHCI under crowded 1 M sucrose environment led us to conclude that in C. reinhardtii FNR also functions as a subunit of PSI-LHCI.


Assuntos
Arabidopsis/metabolismo , Chlamydomonas reinhardtii/enzimologia , Ferredoxina-NADP Redutase/metabolismo , Ferredoxinas/metabolismo , NADP/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema I/metabolismo , Cristalografia por Raios X , Cianobactérias/metabolismo , Transporte de Elétrons , Ferredoxina-NADP Redutase/química , Ferredoxinas/química , Luz , Complexos de Proteínas Captadores de Luz , NADP/química , Complexo de Proteína do Fotossistema I/química , Conformação Proteica
7.
Gene ; 706: 32-42, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31028868

RESUMO

The chloroplastic thioredoxins (Trxs), a family of thiol-disulphide oxidoreductases, are reduced by either ferredoxin (Fd)-dependent Trx reductase (FTR) or reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent Trx reductase (NTR). Two Trx systems are present in chloroplasts including Trxs, Trx-like proteins, and reductase FTR and NTRC. FTR is the main reductant for Trxs in chloroplasts, while the flavoprotein NTRC integrates NTR and Trx activity, and plays multiple roles in the Calvin cycle, the oxidative pentose phosphate pathway (OPPP), anti-peroxidation, tetrapyrrole metabolism, ATP and starch synthesis, and photoperiodic regulation. In addition, not only there exists a reduction potential transfer pathway across the thylakoid membrane, but also FTR and NTRC coordinate with each other to regulate chloroplast redox homeostasis. Herein, we summarise the physiological functions of these two Trx reduction systems, discuss how both regulate redox homeostasis in plant plastids, and emphasize the significance of chloroplast thioredoxin systems in maintaining photosynthetic efficiency in plants.


Assuntos
Cloroplastos/metabolismo , Tiorredoxina Dissulfeto Redutase/fisiologia , Tiorredoxinas/fisiologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cloroplastos/fisiologia , Ferredoxinas/metabolismo , Proteínas com Ferro-Enxofre , Oxirredução , Oxirredutases/metabolismo , Peroxirredoxinas/metabolismo , Fotossíntese/fisiologia , Plastídeos/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo
8.
Nat Commun ; 10(1): 1521, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944323

RESUMO

The response of the prominent marine dinitrogen (N2)-fixing cyanobacteria Trichodesmium to ocean acidification (OA) is critical to understanding future oceanic biogeochemical cycles. Recent studies have reported conflicting findings on the effect of OA on growth and N2 fixation of Trichodesmium. Here, we quantitatively analyzed experimental data on how Trichodesmium reallocated intracellular iron and energy among key cellular processes in response to OA, and integrated the findings to construct an optimality-based cellular model. The model results indicate that Trichodesmium growth rate decreases under OA primarily due to reduced nitrogenase efficiency. The downregulation of the carbon dioxide (CO2)-concentrating mechanism under OA has little impact on Trichodesmium, and the energy demand of anti-stress responses to OA has a moderate negative effect. We predict that if anthropogenic CO2 emissions continue to rise, OA could reduce global N2 fixation potential of Trichodesmium by 27% in this century, with the largest decrease in iron-limiting regions.


Assuntos
Fixação de Nitrogênio/fisiologia , Nitrogênio/metabolismo , Nitrogenase/metabolismo , Trichodesmium/metabolismo , Dióxido de Carbono/metabolismo , Dióxido de Carbono/farmacologia , Simulação por Computador , Metabolismo Energético/efeitos dos fármacos , Ferredoxinas/metabolismo , Concentração de Íons de Hidrogênio , Ferro/metabolismo , Modelos Teóricos , Oceanos e Mares , Água do Mar/química , Água do Mar/microbiologia , Trichodesmium/efeitos dos fármacos , Trichodesmium/enzimologia , Trichodesmium/crescimento & desenvolvimento
9.
Biochemistry ; 58(17): 2228-2242, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30945846

RESUMO

The oxidoreductase YdhV in Escherichia coli has been predicted to belong to the family of molybdenum/tungsten cofactor (Moco/Wco)-containing enzymes. In this study, we characterized the YdhV protein in detail, which shares amino acid sequence homology with a tungsten-containing benzoyl-CoA reductase binding the bis-W-MPT (for metal-binding pterin) cofactor. The cofactor was identified to be of a bis-Mo-MPT type with no guanine nucleotides present, which represents a form of Moco that has not been found previously in any molybdoenzyme. Our studies showed that YdhV has a preference for bis-Mo-MPT over bis-W-MPT to be inserted into the enzyme. In-depth characterization of YdhV by X-ray absorption and electron paramagnetic resonance spectroscopies revealed that the bis-Mo-MPT cofactor in YdhV is redox active. The bis-Mo-MPT and bis-W-MPT cofactors include metal centers that bind the four sulfurs from the two dithiolene groups in addition to a cysteine and likely a sulfido ligand. The unexpected presence of a bis-Mo-MPT cofactor opens an additional route for cofactor biosynthesis in E. coli and expands the canon of the structurally highly versatile molybdenum and tungsten cofactors.


Assuntos
Coenzimas/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Ferredoxinas/química , Metaloproteínas/química , Molibdênio/química , Compostos Organometálicos/química , Oxirredutases/química , Pteridinas/química , Pterinas/química , Coenzimas/genética , Coenzimas/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ferredoxinas/genética , Ferredoxinas/metabolismo , Nucleotídeos de Guanina/química , Nucleotídeos de Guanina/genética , Nucleotídeos de Guanina/metabolismo , Metaloproteínas/genética , Metaloproteínas/metabolismo , Estrutura Molecular , Molibdênio/metabolismo , Compostos Organometálicos/metabolismo , Oxirredução , Oxirredutases/genética , Oxirredutases/metabolismo , Pteridinas/metabolismo , Pterinas/metabolismo
10.
Biochemistry ; 58(18): 2353-2361, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30994334

RESUMO

Cytochrome P450cam is an archetypal example of the vast family of heme monooxygenases and serves as a model for an enzyme that is highly specific for both its substrate and reductase. During catalysis, it undergoes significant conformational changes of the F and G helices upon binding its substrate and redox partner, putidaredoxin (Pdx). Recent studies have shown that Pdx binding to the closed camphor-bound form of ferric P450cam results in its conversion to a fully open state. However, during catalytic turnover, it remains unclear whether this same conformational change also occurs or whether it is coupled to the formation of the critical compound I intermediate. Here, we have examined P450cam bound simultaneously by camphor, CN-, and Pdx as a mimic of the catalytically competent ferrous oxy-P450cam-Pdx state. The combined use of double electron-electron resonance and molecular dynamics showed direct observation of intermediate conformational states of the enzyme upon CN- and subsequent Pdx binding. This state is coupled to the movement of the I helix and residues at the active site, including Arg-186, Asp-251, and Thr-252. These movements enable occupation of a water molecule that has been implicated in proton delivery and peroxy bond cleavage to give compound I. These findings provide a detailed understanding of how the Pdx-induced conformational change may sequentially promote compound I formation followed by product release, while retaining stereoselective hydroxylation of the substrate of this highly specific monooxygenase.


Assuntos
Proteínas de Bactérias/química , Cânfora 5-Mono-Oxigenase/química , Ferredoxinas/química , Simulação de Dinâmica Molecular , Conformação Proteica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Cânfora 5-Mono-Oxigenase/genética , Cânfora 5-Mono-Oxigenase/metabolismo , Domínio Catalítico , Ferredoxinas/metabolismo , Oxirredução , Ligação Proteica , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Especificidade por Substrato
11.
mBio ; 10(2)2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30837343

RESUMO

Control of electron flux is critical in both natural and bioengineered systems to maximize energy gains. Both small molecules and proteins shuttle high-energy, low-potential electrons liberated during catabolism through diverse metabolic landscapes. Ferredoxin (Fd) proteins-an abundant class of Fe-S-containing small proteins-are essential in many species for energy conservation and ATP production strategies. It remains difficult to model electron flow through complicated metabolisms and in systems in which multiple Fd proteins are present. The overlap of activity and/or limitations of electron flux through each Fd can limit physiology and metabolic engineering strategies. Here we establish the interplay, reactivity, and physiological role(s) of the three ferredoxin proteins in the model hyperthermophile Thermococcus kodakarensis We demonstrate that the three loci encoding known Fds are subject to distinct regulatory mechanisms and that specific Fds are utilized to shuttle electrons to separate respiratory and energy production complexes during different physiological states. The results obtained argue that unique physiological roles have been established for each Fd and that continued use of T. kodakarensis and related hydrogen-evolving species as bioengineering platforms must account for the distinct Fd partnerships that limit flux to desired electron acceptors. Extrapolating our results more broadly, the retention of multiple Fd isoforms in most species argues that specialized Fd partnerships are likely to influence electron flux throughout biology.IMPORTANCE High-energy electrons liberated during catabolic processes can be exploited for energy-conserving mechanisms. Maximal energy gains demand these valuable electrons be accurately shuttled from electron donor to appropriate electron acceptor. Proteinaceous electron carriers such as ferredoxins offer opportunities to exploit specific ferredoxin partnerships to ensure that electron flux to critical physiological pathways is aligned with maximal energy gains. Most species encode many ferredoxin isoforms, but very little is known about the role of individual ferredoxins in most systems. Our results detail that ferredoxin isoforms make largely unique and distinct protein interactions in vivo and that flux through one ferredoxin often cannot be recovered by flux through a different ferredoxin isoform. The results obtained more broadly suggest that ferredoxin isoforms throughout biological life have evolved not as generic electron shuttles, but rather serve as selective couriers of valuable low-potential electrons from select electron donors to desirable electron acceptors.


Assuntos
Transporte de Elétrons , Metabolismo Energético , Ferredoxinas/metabolismo , Thermococcus/enzimologia , Thermococcus/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação da Expressão Gênica em Archaea , Thermococcus/genética
12.
Nature ; 566(7744): 411-414, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30742075

RESUMO

Cyclic electron flow around photosystem I (PSI) is a mechanism by which photosynthetic organisms balance the levels of ATP and NADPH necessary for efficient photosynthesis1,2. NAD(P)H dehydrogenase-like complex (NDH) is a key component of this pathway in most oxygenic photosynthetic organisms3,4 and is the last large photosynthetic membrane-protein complex for which the structure remains unknown. Related to the respiratory NADH dehydrogenase complex (complex I), NDH transfers electrons originating from PSI to the plastoquinone pool while pumping protons across the thylakoid membrane, thereby increasing the amount of ATP produced per NADP+ molecule reduced4,5. NDH possesses 11 of the 14 core complex I subunits, as well as several oxygenic-photosynthesis-specific (OPS) subunits that are conserved from cyanobacteria to plants3,6. However, the three core complex I subunits that are involved in accepting electrons from NAD(P)H are notably absent in NDH3,5,6, and it is therefore not clear how NDH acquires and transfers electrons to plastoquinone. It is proposed that the OPS subunits-specifically NdhS-enable NDH to accept electrons from its electron donor, ferredoxin3-5,7. Here we report a 3.1 Å structure of the 0.42-MDa NDH complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, obtained by single-particle cryo-electron microscopy. Our maps reveal the structure and arrangement of the principal OPS subunits in the NDH complex, as well as an unexpected cofactor close to the plastoquinone-binding site in the peripheral arm. The location of the OPS subunits supports a role in electron transfer and defines two potential ferredoxin-binding sites at the apex of the peripheral arm. These results suggest that NDH could possess several electron transfer routes, which would serve to maximize plastoquinone reduction and avoid deleterious off-target chemistry of the semi-plastoquinone radical.


Assuntos
Microscopia Crioeletrônica , Cianobactérias/química , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/ultraestrutura , NADPH Desidrogenase/química , NADPH Desidrogenase/ultraestrutura , Oxigênio/metabolismo , Fotossíntese , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Coenzimas/química , Coenzimas/metabolismo , Cianobactérias/enzimologia , Transporte de Elétrons , Complexo I de Transporte de Elétrons/metabolismo , Ferredoxinas/metabolismo , Modelos Biológicos , Modelos Moleculares , NADPH Desidrogenase/metabolismo , Oxirredução , Complexo de Proteína do Fotossistema I/metabolismo , Plastoquinona/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
13.
Biochim Biophys Acta Gen Subj ; 1863(4): 651-660, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30639162

RESUMO

BACKGROUND: Ferredoxins are small iron-sulfur proteins that participate as electron donors in various metabolic pathways. They are recognized substrates of ferredoxin-NADP+ reductases (FNR) in redox metabolisms in mitochondria, plastids, and bacteria. We previously found a plastidic-type FNR in Leptospira interrogans (LepFNR), a parasitic bacterium of animals and humans. Nevertheless, we did not identify plant-type ferredoxins or flavodoxins, the common partners of this kind of FNR. METHODS: Sequence alignment, phylogenetical analyses and structural modeling were performed for the identification of a 2[4Fe4S] ferredoxin (LepFd2) as a putative redox partner of LepFNR in L. interrogans. The gene encoding LepFd2 was cloned and the protein overexpressed and purified. The functional properties of LepFd2 and LepFNR-LepFd2 complex were analyzed by kinetic and mutagenesis studies. RESULTS: We succeeded in expressing and purifying LepFd2 with its FeS cluster properly bound. We found that LepFd2 exchanges electrons with LepFNR. Moreover, a unique structural subdomain of LepFNR (loop P75-Y91), was shown to be involved in the recognition and binding of LepFd2. This structural subdomain is not found in other FNR homologs. CONCLUSIONS: We report for the first time a redox pair in L. interrogans in which a plastidic FNR exchanges electron with a bacterial 2[4Fe4S] ferredoxin. We characterized this reaction and proposed a model for the productive LepFNR-LepFd2 complex. GENERAL SIGNIFICANCE: Our findings suggest that the interaction of LepFNR with the iron-sulfur protein would be different from the one previously described for the homolog enzymes. This knowledge would be useful for the design of specific LepFNR inhibitors.


Assuntos
Ferredoxina-NADP Redutase/metabolismo , Ferredoxinas/metabolismo , Leptospira interrogans/enzimologia , Sequência de Aminoácidos , Ferredoxina-NADP Redutase/química , Ferredoxinas/química , Modelos Moleculares , Oxirredução , Filogenia , Conformação Proteica , Alinhamento de Sequência
14.
Metab Eng ; 51: 32-42, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30218716

RESUMO

The thermophilic anaerobes Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are good candidates for lignocellulosic ethanol production. T. saccharolyticum has been successfully engineered to produce ethanol at high titer (70 g/L). The maximum ethanol titer of engineered strains of C. thermocellum is only 25 g/L. We hypothesize that one or more of the enzymes in the ethanol production pathway in C. thermocellum is not adequate for ethanol production at high titer. In this study, we focused on the enzymes responsible for the part of the ethanol production pathway from pyruvate to ethanol. In T. saccharolyticum, we replaced all of the genes encoding proteins in this pathway with their homologs from C. thermocellum and examined what combination of gene replacements restricted ethanol titer. We found that a pathway consisting of Ct_nfnAB, Ct_fd, Ct_adhE and Ts_pforA was sufficient to support ethanol titer greater than 50 g/L, however replacement of Ts_pforA by Ct_pfor1 dramatically decreased the maximum ethanol titer to 14 g/L. We then demonstrated that the reason for reduced ethanol production is that the Ct_pfor1 is inhibited by accumulation of ethanol and NADH, while Ts_pforA is not.


Assuntos
Álcool Desidrogenase/metabolismo , Aldeído Desidrogenase/metabolismo , Clostridium thermocellum/metabolismo , Ferredoxinas/metabolismo , NADH NADPH Oxirredutases/metabolismo , Piruvato Sintase/metabolismo , Thermoanaerobacterium/metabolismo , Álcool Desidrogenase/genética , Aldeído Desidrogenase/genética , Clostridium thermocellum/genética , Fermentação , Ferredoxinas/genética , Engenharia Metabólica , NADH NADPH Oxirredutases/genética , Plasmídeos/genética
15.
J Microbiol Biotechnol ; 29(1): 44-54, 2019 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-30415526

RESUMO

Geldanamycin and its derivatives, inhibitors of heat shock protein 90, are considered potent anticancer drugs, although their biosynthetic pathways have not yet been fully elucidated. The key step of conversion of 4,5-dihydrogeldanamycin to geldanamycin was expected to catalyze by a P450 monooxygenase, Gel16. The adequate bioconversions by cytochrome P450 mostly rely upon its interaction with redox partners. Several ferredoxin and ferredoxin reductases are available in the genome of certain organisms, but only a few suitable partners can operate in full efficiency. In this study, we have expressed cytochrome P450 gel16 in Escherichia coli and performed an in vitro assay using 4,5-dihydrogeldanamycin as a substrate. We demonstrated that the in silico method can be applicable for the efficient mining of convenient endogenous redox partners (9 ferredoxins and 6 ferredoxin reductases) against CYP Gel16 from Streptomyces hygroscopicus. The distances for ligand FDX4-FDR6 were found to be 9.384 Å. Similarly, the binding energy between Gel16-FDX4 and FDX4-FDR6 were -611.88 kcal/mol and -834.48 kcal/mol, respectively, suggesting the lowest distance and binding energy rather than other redox partners. These findings suggest that the best redox partners of Gel16 could be NADPH → FDR6 → FDX4 → Gel16.


Assuntos
Proteínas de Bactérias/metabolismo , Benzoquinonas/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Transporte de Elétrons , Ferredoxinas/metabolismo , Lactamas Macrocíclicas/metabolismo , NADP/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Vias Biossintéticas , Clonagem Molecular , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Simulação de Acoplamento Molecular , Oxirredução , Ligação Proteica , Streptomyces/genética , Streptomyces/metabolismo
16.
Protein Sci ; 28(1): 257-266, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30418685

RESUMO

Most organisms contain multiple soluble protein-based redox carriers such as members of the ferredoxin (Fd) family, that contain one or more iron-sulfur clusters. The potential redundancy of Fd proteins is poorly understood, particularly in connection to the ability of Fd proteins to deliver reducing equivalents to members of the "radical SAM," or S-adenosylmethionine radical enzyme (ARE) superfamily, where the activity of all known AREs requires that an essential iron-sulfur cluster bound by the enzyme be reduced to the catalytically relevant [Fe4 S4 ]1+ oxidation state. As it is still unclear whether a single Fd in a given organism is specific to individual redox partners, we have examined the five Fd proteins found within Thermotoga maritima via direct electrochemistry, to compare them in a side-by-side fashion for the first time. While a single [Fe4 S4 ]-cluster bearing Fd (TM0927) has a potential of -420 mV, the other four 2x[Fe4 S4 ]-bearing Fds (TM1175, TM1289, TM1533, and TM1815) have potentials that vary significantly, including cases where the two clusters of the same Fd are essentially coincident (e.g., TM1175) and those where the potentials are well separate (TM1815).


Assuntos
Ferredoxinas/química , Thermotoga maritima/química , Ferredoxinas/metabolismo , Oxirredução , Thermotoga maritima/metabolismo
17.
Protein Sci ; 28(1): 267-282, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30394621

RESUMO

MiaB is a member of the methylthiotransferase subclass of the radical S-adenosylmethionine (SAM) superfamily of enzymes, catalyzing the methylthiolation of C2 of adenosines bearing an N6 -isopentenyl (i6 A) group found at position 37 in several tRNAs to afford 2-methylthio-N6 -(isopentenyl)adenosine (ms2 i6 A). MiaB uses a reduced [4Fe-4S]+ cluster to catalyze a reductive cleavage of SAM to generate a 5'-deoxyadenosyl 5'-radical (5'-dA•)-a required intermediate in its reaction-as well as an additional [4Fe-4S]2+ auxiliary cluster. In Escherichia coli and many other organisms, re-reduction of the [4Fe-4S]2+ cluster to the [4Fe-4S]+ state is accomplished by the flavodoxin reducing system. Most mechanistic studies of MiaBs have been carried out on the enzyme from Thermotoga maritima (Tm), which lacks the flavodoxin reducing system, and which is not activated by E. coli flavodoxin. However, the genome of this organism encodes five ferredoxins (TM0927, TM1175, TM1289, TM1533, and TM1815), each of which might donate the requisite electron to MiaB and perhaps to other radical SAM enzymes. The genes encoding each of these ferredoxins were cloned, and the associated proteins were isolated and shown to support turnover by Tm MiaB. In addition, TM1639, the ferredoxin-NADP+ oxidoreductase subunit α (NfnA) from Tm was overproduced and isolated and shown to provide electrons to the Tm ferredoxins during Tm MiaB turnover. The resulting reactions demonstrate improved coupling between formation of the 5'-dA• and ms2 i6 A production, indicating that only one hydrogen atom abstraction is required for the reaction.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Sulfurtransferases/metabolismo , Thermotoga maritima/enzimologia , Transporte de Elétrons/fisiologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Ferredoxinas/genética , Ferredoxinas/metabolismo , Sulfurtransferases/genética , Thermotoga maritima/genética
18.
Int J Biol Macromol ; 122: 636-643, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30391427

RESUMO

The exposed hydrophobic patches of protein are widely detected through the binding by the fluorescent probes such as 1-anilino-8-naphthalene sulfonate (ANS), Nile Red (NR) and 1-(N-phenylamino) naphthalene, N-(1-Naphthyl) aniline (1NPN). Interestingly, at pH4, where the Toxoplasma gondii Ferredoxin-NADP(+) reductase (TgFNR) is stable, an exclusive binding and fluorescence emission was observed for ANS. To understand the underlying difference in the binding of ANS, NR and 1NPN; their effect on the protein structure was studied in detail. ANS was found to interact with TgFNR via electrostatic as well as hydrophobic interactions at pH4. NR and 1NPN did not show any such binding to TgFNR in the similar conditions, however showed strong hydrophobic interaction in the presence of NaCl or DSS (2, 2-dimethyl-2-silapentane-5-sulfonate). The subsequent structural studies suggest that ANS, NaCl and DSS induced partial unfolding of TgFNR by modulating ionic interactions of the enzyme, leading to the exposure of buried hydrophobic patches amicable for the binding by NR and 1NPN. The induced unfolding of TgFNR by ANS is unique and thus cautions to use the fluorescent dye as simple indicator to probe the exposed hydrophobic patches of the protein or its folding intermediates.


Assuntos
Naftalenossulfonato de Anilina/metabolismo , Naftalenossulfonato de Anilina/farmacologia , Ferredoxinas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , NADP/metabolismo , Oxirredutases/metabolismo , Toxoplasma/enzimologia , Oxirredutases/química , Ligação Proteica , Desdobramento de Proteína/efeitos dos fármacos
19.
Appl Environ Microbiol ; 85(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30552192

RESUMO

In Liberibacter asiaticus, PrbP is an important transcriptional accessory protein that regulates gene expression through interactions with the RNA polymerase ß-subunit and a specific sequence on the promoter region. The constitutive expression of prbP observed upon chemical inactivation of PrbP-DNA interactions in vivo indicated that the expression of prbP was not autoregulated at the level of transcription. This observation suggested that a modulatory mechanism via protein-protein interactions may be involved. In silico genome association analysis identified FerR (CLIBASIA_01505), a putative ferredoxin-like protein, as a PrbP-interacting protein. Using a bacterial two-hybrid system and immunoprecipitation assays, interactions between PrbP and FerR were confirmed. In vitro transcription assays were used to show that FerR can increase the activity of PrbP by 16-fold when present in the PrbP-RNA polymerase reaction mixture. The FerR protein-protein interaction surface was predicted by structural modeling and followed by site-directed mutagenesis. Amino acids V20, V23, and C40 were identified as the most important residues in FerR involved in the modulation of PrbP activity in vitro The regulatory mechanism of FerR abundance was examined at the transcription level. In contrast to prbP of L. asiaticus (prbP Las), mRNA levels of ferR of L. asiaticus (ferR Las) are induced by an increase in osmotic pressure. The results of this study revealed that the activity of the transcriptional activator PrbPLas is modulated via interactions with FerRLas The induction of ferR Las expression by osmolarity provides insight into the mechanisms of adjusting gene expression in response to host environmental signals in L. asiaticus IMPORTANCE The rapid spread and aggressive progression of huanglongbing (HLB) in the major citrus-producing areas have raised global recognition of and vigilance to this disease. As a result, the causative agent, Liberibacter asiaticus, has been investigated from various perspectives. However, gene expression regulatory mechanisms that are important for the survival and persistence of this intracellular pathogen remain largely unexplored. PrbP is a transcriptional accessory protein important for L. asiaticus survival in the plant host. In this study, we investigated the interactions between PrbP in L. asiaticus (PrbPLas) and a ferredoxin-like protein (FerR) in L. asiaticus, FerRLas We show that the presence of FerR stabilizes and augments the activity of PrbPLas In addition, we demonstrate that the expression of ferR is induced by increases in osmolarity in Liberibacter crescens Altogether, these results suggest that FerRLas and PrbPLas may play important roles in the regulation of gene expression in response to changing environmental signals during L. asiaticus infection in the citrus host.


Assuntos
Ferredoxinas/genética , Ferredoxinas/metabolismo , Rhizobiaceae/genética , Rhizobiaceae/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citrus/microbiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno , Modelos Moleculares , Pressão Osmótica , Doenças das Plantas/microbiologia , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas
20.
Proc Natl Acad Sci U S A ; 115(51): E12111-E12120, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30514818

RESUMO

Iron chronically limits aquatic photosynthesis, especially in marine environments, and the correct perception and maintenance of iron homeostasis in photosynthetic bacteria, including cyanobacteria, is therefore of global significance. Multiple adaptive mechanisms, responsive promoters, and posttranscriptional regulators have been identified, which allow cyanobacteria to respond to changing iron concentrations. However, many factors remain unclear, in particular, how iron status is perceived within the cell. Here we describe a cyanobacterial ferredoxin (Fed2), with a unique C-terminal extension, that acts as a player in iron perception. Fed2 homologs are highly conserved in photosynthetic organisms from cyanobacteria to higher plants, and, although they belong to the plant type ferredoxin family of [2Fe-2S] photosynthetic electron carriers, they are not involved in photosynthetic electron transport. As deletion of fed2 appears lethal, we developed a C-terminal truncation system to attenuate protein function. Disturbed Fed2 function resulted in decreased chlorophyll accumulation, and this was exaggerated in iron-depleted medium, where different truncations led to either exaggerated or weaker responses to low iron. Despite this, iron concentrations remained the same, or were elevated in all truncation mutants. Further analysis established that, when Fed2 function was perturbed, the classical iron limitation marker IsiA failed to accumulate at transcript and protein levels. By contrast, abundance of IsiB, which shares an operon with isiA, was unaffected by loss of Fed2 function, pinpointing the site of Fed2 action in iron perception to the level of posttranscriptional regulation.


Assuntos
Ferredoxinas/fisiologia , Ferro/metabolismo , Fotossíntese/fisiologia , Synechocystis/fisiologia , Adaptação Fisiológica , Clorofila/metabolismo , Ferredoxinas/química , Ferredoxinas/metabolismo , Homeostase/genética , Synechocystis/genética , Synechocystis/metabolismo
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