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1.
Microbiol Res ; 282: 127661, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38432016

RESUMO

In yeasts, ferric reductase catalyzes reduction of ferric ion to ferrous form, which is essential for the reductive iron assimilation system. However, the physiological roles of ferric reductases remain largely unknown in the filamentous fungi. In this study, genome-wide annotation revealed thirteen ferric reductase-like (Fre) proteins in the filamentous insect pathogenic fungus Beauveria bassiana, and all their functions were genetically characterized. Ferric reductase family proteins exhibit different sub-cellular distributions (e.g., cell periphery and vacuole), which was due to divergent domain architectures. Fre proteins had a synergistic effect on fungal virulence, which was ascribed to their distinct functions in different physiologies. Ten Fre proteins were not involved in reduction of ferric ion in submerged mycelia, but most proteins contributed to blastospore development. Only two Fre proteins significantly contributed to B. bassiana vegetative growth under the chemical-induced iron starvation, but most Fre proteins were involved in resistance to osmotic and oxidative stresses. Notably, a bZIP-type transcription factor HapX bound to the promoter regions of all FRE genes in B. bassiana, and displayed varying roles in the transcription activation of these genes. This study reveals the important role of BbFre family proteins in development, stress response, and insect pathogenicity, as well as their distinctive role in the absorption of ferric iron from the environment.


Assuntos
Beauveria , FMN Redutase , Animais , Virulência/genética , Beauveria/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Esporos Fúngicos , Insetos , Ferro/metabolismo
2.
Biochemistry ; 62(18): 2751-2762, 2023 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-37651343

RESUMO

The FMN reductases (SsuE and MsuE of the alkanesulfonate monooxygenase systems) supply reduced flavin to their partner monooxygenases for the desulfonation of alkanesulfonates. Flavin reductases that comprise two-component systems must be able to regulate both flavin reduction and transfer. One mechanism to control these distinct processes is through changes in the oligomeric state of the enzymes. Despite their similar overall structures, SsuE and MsuE showed clear differences in their oligomeric states in the presence of substrates. The oligomeric state of SsuE was converted from a tetramer to a dimer/tetramer equilibrium in the presence of FMN or NADPH in analytical ultracentrifugation studies. Conversely, MsuE shifted from a dimer to a single tetrameric state with FMN, and the NADPH substrate did not induce a similar oligomeric shift. There was a fast tetramer to dimer equilibrium shift occurring at the dimer/dimer interface in H/D-X investigations with apo SsuE. Formation of the SsuE/FMN complex slowed the tetramer/dimer conversion, leading to a slower exchange along the dimer/dimer interface. The oligomeric shift of the MsuE/FMN complex from a dimer to a distinct tetramer showed a decrease in H/D-X in the region around the π-helices at the dimer/dimer interface. Both SsuE and MsuE showed a comparable and significant increase in the melting temperature with the addition of FMN, indicating the conformers formed by each FMN-bound enzyme had increased stability. A mechanism that supports the different structural shifts is rationalized by the different roles these enzymes play in providing reduced flavin to single or multiple monooxygenase enzymes.


Assuntos
FMN Redutase , Compostos Orgânicos , NADP , FMN Redutase/genética , Flavinas , Oxigenases de Função Mista/genética , Polímeros , Enxofre
3.
Methods Mol Biol ; 2665: 31-36, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37166590

RESUMO

A sensitive FerroZine assay is used to measure the membrane-bound ferric-chelate reductase activity in the Arabidopsis thaliana roots. In Arabidopsis, FRO2 (FERRIC CHELATE REDUCTASE 2) encodes the Fe(III) chelate reductase and its expression is induced by iron deficiency. As FRO2 reduces Fe(III) to soluble Fe(II), the resulting Fe(II) forms a purple-colored complex with the dye FerroZine. The concentration of the Fe(II)-FerroZine is directly proportional to the absorbance at 562 nm.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , FMN Redutase/genética , FMN Redutase/metabolismo , Compostos Férricos/metabolismo , Ferrozina/metabolismo , Proteínas de Arabidopsis/metabolismo , Compostos Ferrosos , Raízes de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas
4.
Elife ; 112022 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-36200752

RESUMO

Heme can serve as iron source in many environments, including the iron-poor animal host environment. The fungal pathobiont Candida albicans expresses a family of extracellular CFEM hemophores that capture heme from host proteins and transfer it across the cell wall to the cell membrane, to be endocytosed and utilized as heme or iron source. Here, we identified Frp1 and Frp2, two ferric reductase (FRE)-related proteins that lack an extracellular N-terminal substrate-binding domain, as being required for hemoglobin heme utilization and for sensitivity to toxic heme analogs. Frp1 and Frp2 redistribute to the plasma membrane in the presence of hemin, consistent with a direct role in heme trafficking. Expression of Frp1 with the CFEM hemophore Pga7 can promote heme utilization in Saccharomyces cerevisiae as well, confirming the functional interaction between these proteins. Sequence and structure comparison reveals that the CFEM hemophores are related to the FRE substrate-binding domain that is missing in Frp1/2. We conclude that Frp1/2 and the CFEM hemophores form a functional complex that evolved from FREs to enable extracellular heme uptake.


Hosts and disease-causing fungi are often locked into a battle over resources. The host will attempt to withhold molecules that the fungus needs to survive, while the pathogen will try to find alternative routes to obtain them. Candida albicans, for example, can go after the atoms of iron embedded in the proteins of the organism it infects. To do so it releases molecules known as hemophores, which scavenge the iron-containing heme molecule that equips oxygen-carrying proteins in the blood. Once captured, the heme is carried across the wall that protects C. albicans from the environment and brought to the membrane of the cell. It is then taken in and trafficked inside vesicles to its destination. However, the identity of the molecular actors which help to bridge the internal and external segments of the heme journey remain unclear. Previous studies have shown that the hemophore Pga7 is involved, but this protein is attached to the outside of the cell membrane, where it cannot directly interact with the import machinery. Roy et al. set out to discover this missing link. Examining the genomes of fungal species related to C. albicans highlighted two membrane proteins, Frp1 and Frp2, which could participate in heme uptake. Protein sequence comparison revealed that Frp1 and Frp2 were closely related to ferric reductases, a group of membrane enzymes which can chemically alter extracellular iron prior to uptake. Deleting the genes for Frp1 and Frp2 rendered C. albicans cells incapable of taking in heme. Conversely, a fungal species which cannot normally uptake heme could efficiently internalise these complexes when artificially equipped with Frp1 and Pga7, suggesting that the two proteins work closely together. Finally, protein structure comparisons highlighted that an extracellular domain present in ferric reductases but absent in Frp1 and Frp2 is, in fact, related to Pga7 and other hemophores. This implies that the iron and heme uptake systems may share a common evolutionary origin. Overall, the work by Roy et al. reveals a new family of proteins which allow disease-causing fungi to steal iron from their hosts. This knowledge may be useful to design better anti-fungal treatments.


Assuntos
Candida albicans , FMN Redutase , Animais , FMN Redutase/metabolismo , Candida albicans/genética , Candida albicans/metabolismo , Heme/metabolismo , Ferro/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo
5.
Microb Pathog ; 172: 105779, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36116609

RESUMO

Nicotinamide Adenine Dinucleotide-Dependent (NADH) flavin oxidoreductase and NADH oxidase (NOX) are important virulence factors of Mycoplasma hyopneumoniae (Mhp), which are devoted to the function of adhesion, oxidative stress damage and apoptosis to host cells in our previous studies. Here, immune responses of NADH flavin oxidoreductase (NFOR) and NOX in mice and immune efficacy inoculated with intramuscular (IM), intranasal (IN), intramuscular unite intranasal (IM + IN) approaches were evaluated and compared. Cellular immunity levels, systemic immune and local mucosal immune responses were investigated by indirect enzyme-linked immunosorbent assay (iELISA) and quantitative reverse transcription PCR (qRT-PCR). Mice inoculated with NFOR and NOX by IM and IN or IM + IN could induce obvious secretion of specific immunoglobulin G (IgG) and secretory immunoglobulin A antibodies (sIgA) compared to those in negative control group. IM + IN inoculation resulted in systemic and local mucosal immune responses that were strongly produced. Moreover, Mhp NFOR and NOX could activate local mucosal immune responses mediated by Th1 and Th17 cells by IN. Our finding supported the notion that IM + IN was an effective immunization route for Mhp, which lays a foundation for more effective prevention of Mhp, and provides theoretical basis for the development of new subunit vaccines of Mhp.


Assuntos
Mycoplasma hyopneumoniae , Camundongos , Animais , Imunidade nas Mucosas , NAD , Fatores de Virulência , Células Th17 , FMN Redutase , Vacinas Bacterianas , Imunoglobulina G , Vacinas de Subunidades , Imunoglobulina A Secretora , Flavinas , Camundongos Endogâmicos BALB C
6.
Int J Mol Sci ; 23(18)2022 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-36142217

RESUMO

Magnetosomes of magnetotactic bacteria consist of magnetic nanocrystals with defined morphologies enclosed in vesicles originated from cytoplasmic membrane invaginations. Although many proteins are involved in creating magnetosomes, a single magnetosome protein, Mms6 from Magnetospirillum magneticum strain AMB-1, can direct the crystallization of magnetite nanoparticles in vitro. The in vivo role of Mms6 in magnetosome formation is debated, and the observation that Mms6 binds Fe3+ more tightly than Fe2+ raises the question of how, in a magnetosome environment dominated by Fe3+, Mms6 promotes the crystallization of magnetite, which contains both Fe3+ and Fe2+. Here we show that Mms6 is a ferric reductase that reduces Fe3+ to Fe2+ using NADH and FAD as electron donor and cofactor, respectively. Reductase activity is elevated when Mms6 is integrated into either liposomes or bicelles. Analysis of Mms6 mutants suggests that the C-terminal domain binds iron and the N-terminal domain contains the catalytic site. Although Mms6 forms multimers that involve C-terminal and N-terminal domain interactions, a fusion protein with ubiquitin remains a monomer and displays reductase activity, which suggests that the catalytic site is fully in the monomer. However, the quaternary structure of Mms6 appears to alter the iron binding characteristics of the C-terminal domain. These results are consistent with a hypothesis that Mms6, a membrane protein, promotes the formation of magnetite in vivo by a mechanism that involves reducing iron.


Assuntos
Magnetossomos , Magnetospirillum , Proteínas de Bactérias/química , FMN Redutase/metabolismo , Óxido Ferroso-Férrico/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Ferro/metabolismo , Lipídeos/análise , Lipossomos/metabolismo , Magnetossomos/metabolismo , Magnetospirillum/metabolismo , Proteínas de Membrana/metabolismo , NAD/metabolismo , Ubiquitinas/metabolismo
7.
Eur J Cell Biol ; 101(3): 151230, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35550931

RESUMO

Iron is the most abundant transition metal in all living organisms and is essential for several cellular activities, including respiration, oxygen transport, energy production and regulation of gene expression. Iron starvation is used by professional phagocytes, from Dictyostelium to macrophages, as a form of defense mechanism against intracellular pathogens. Previously, we showed that Dictyostelium cells express the proton-driven iron transporter Nramp1 (Natural Resistance-Associated Macrophage Protein 1) and the homolog NrampB (Nramp2) in membranes of macropinosomes and phagosomes or of the contractile vacuole network, respectively. The Nramp-driven transport of iron across membranes is selective for ferrous ions. Since iron is mostly present as ferric ions in growth media and in engulfed bacteria, we have looked for proteins with ferric reductase activity. The Dictyostelium genome does not encode for classical STEAP (Six-Transmembrane Epithelial Antigen of Prostate) ferric reductases, but harbors three genes encoding putative ferric chelate reductase belonging to the Cytochrome b561 family containing a N terminus DOMON domain (DOpamine ß-MONooxygenase N-terminal domain). We have cloned the three genes, naming them fr1A, fr1B and fr1C. fr1A and fr1B are mainly expressed in the vegetative stage while fr1C is highly expressed in the post aggregative stage. All three reductases are localized in the endoplasmic reticulum, but Fr1A is also found in endolysosomal vesicles, in the Golgi and, to a much lower degree, in the plasma membrane, whereas Fr1C is homogeneously distributed in the plasma membrane and in macropinosomal and phagosomal membranes. To gain insight in the function of the three genes we generated KO mutants, but gene disruption was successful only for two of them (fr1A and fr1C), being very likely lethal for fr1B. fr1A- shows a slight delay in the aggregation stage of development, while fr1C- gives rise to large multi-tipped streams during aggregation and displays a strong delay in fruiting body formation. The two single mutants display altered cell growth under conditions of ferric ions overloading and, in the ability to reduce Fe3+, confirming a role of these putative ferric reductases in iron reduction and transport from endo-lysosomal vesicles to the cytosol.


Assuntos
Dictyostelium , FMN Redutase , Dictyostelium/enzimologia , Dictyostelium/genética , FMN Redutase/genética , FMN Redutase/metabolismo , Íons/metabolismo , Ferro/metabolismo
8.
Appl Environ Microbiol ; 88(11): e0051922, 2022 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-35612301

RESUMO

Bacteria coping with oxygen deficiency use alternative terminal electron acceptors for NADH regeneration, particularly fumarate. Fumarate is reduced by the FAD_binding_2 domain of cytoplasmic fumarate reductase in many bacteria. The variability of the primary structure of this domain in homologous proteins suggests the existence of reducing activities with different specificities. Here, we produced and characterized one such protein encoded in the Vibrio harveyi genome (GenBank ID: AIV07243) and found it to be a specific NADH:acrylate oxidoreductase (ARD). This previously unknown enzyme is formed by the OYE-like, FMN_bind, and FAD_binding_2 domains and contains covalently bound flavin mononucleotide (FMN) and noncovalently bound flavin adenine dinucleotide (FAD) and FMN in a ratio of 1:1:1. The covalently bound FMN is absolutely required for activity and is attached by the specific flavin transferase, ApbE, to the FMN_bind domain. Quantitative reverse transcription PCR (RT-qPCR) and activity measurements indicated dramatic stimulation of ARD biosynthesis by acrylate in the V. harveyi cells grown aerobically. In contrast, the ard gene expression in the cells grown anaerobically without acrylate was higher than that in aerobic cultures and increased only 2-fold in the presence of acrylate. These findings suggest that the principal role of ARD in Vibrio is energy-saving detoxification of acrylate coming from the environment. IMPORTANCE The benefits of the massive genomic information accumulated in recent years for biological sciences have been limited by the lack of data on the function of most gene products. Approximately half of the known prokaryotic genes are annotated as "proteins with unknown functions," and many other genes are annotated incorrectly. Thus, the functional and structural characterization of the products of such genes, including identification of all existing enzymatic activities, is a pressing issue in modern biochemistry. In this work, we have shown that the product of the V. harveyi ard gene exhibits a yet-undescribed NADH:acrylate oxidoreductase activity. This activity may allow acrylate detoxification and its use as a terminal electron acceptor in anaerobic or substrate in aerobic respiration of marine and other bacteria.


Assuntos
Mononucleotídeo de Flavina , Vibrio , Acrilatos , Sequência de Aminoácidos , FMN Redutase/metabolismo , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Fumaratos , NAD/metabolismo , NADH Desidrogenase/metabolismo , NADH NADPH Oxirredutases/metabolismo , Vibrio/metabolismo
9.
Cells ; 10(11)2021 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-34831377

RESUMO

Hydrangea macrophylla is a popular perennial ornamental shrub commercially grown as potted plants, landscape plants, and cut flowers. In the process of reproduction and production of ornamental plants, the absorption of nutrients directly determines the value of the ornamental plants. Hydrangea macrophylla is very sensitive to the content and absorption of the micronutrient iron (Fe) that affects growth of its shoots. However, the physiological activity of Fe as affected by deficiency or supplementation is unknown. This work aimed at preliminary exploring the relationship between Fe and photosynthesis, and also to find the most favorable iron source and level of pH for the growth of H. macrophylla. Two Fe sources, non-chelated iron sulfate (FeSO4) and iron ethylenediaminetetraacetic acid (Fe-EDTA), were supplemented to the multipurpose medium with a final Fe concentration of 2.78 mg·L-1. The medium without any Fe supplementation was used as the control. The pH of the agar-solidified medium was adjusted to either 4.70, 5.70, or 6.70, before autoclaving. The experiment was conducted in a culture room for 60 days with 25/18 °C day and night temperatures, and a 16-hour photoperiod provided at a light intensity of 50 mmol·m-2·s-1 photosynthetic photon flux density (PPFD) from white light-emitting diodes. Supplementary Fe increased the tissue Fe content, and leaves were greener with the medium pH of 4.70, regardless of the Fe source. Compared to the control, the number of leaves for plantlets treated with FeSO4 and Fe-EDTA were 2.0 and 1.5 times greater, respectively. The chlorophyll, macronutrient, and micronutrient contents were the greatest with Fe-EDTA at pH 4.70. Furthermore, the Fe in the leaf affected the photosynthesis by regulating stomata development, pigment content, and antioxidant system, and also by adjusting the expression of genes related to Fe absorption, transport, and redistribution. Supplementation of Fe in a form chelated with EDTA along with a medium pH of 4.70 was found to be the best for the growth and development of H. macrophylla plantlets cultured in vitro.


Assuntos
Hydrangea/crescimento & desenvolvimento , Ferro/farmacologia , Antioxidantes/metabolismo , Proteínas de Arabidopsis/genética , Sequência de Bases , FMN Redutase/metabolismo , Fluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hydrangea/anatomia & histologia , Hydrangea/efeitos dos fármacos , Hydrangea/enzimologia , Concentração de Íons de Hidrogênio , Micronutrientes/análise , Modelos Biológicos , Nutrientes/análise , Fotossíntese/efeitos dos fármacos , Pigmentação/efeitos dos fármacos , Pigmentos Biológicos/metabolismo , Proteínas de Plantas/genética , Estômatos de Plantas/efeitos dos fármacos , Estômatos de Plantas/fisiologia , Estômatos de Plantas/ultraestrutura , Solubilidade
10.
BMC Microbiol ; 21(1): 319, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34798816

RESUMO

BACKGROUND: Tuberculosis (TB) remains an important public health problem since it is the major cause of elevated morbidity and mortality globally. Previous works have shown that Mycobacterium tuberculosis (Mtb); the prime causative agent of the deadly disease has dormancy survival regulator (DosR) regulon, a two-component regulatory system which controls the transcription of more than 50 genes. However, the structure and detailed functions of these DosR regulated genes are largely undetermined. Out of many DosR regulon genes, Rv3131 gets up regulated in hypoxic conditions and was believed to encode for a nitroreductase flavoprotein. The utilization of mycobacteria-specific model systems has greatly added to our understanding of the molecular mechanisms involved in the life cycle and pathogenesis of Mtb. RESULTS: In this study the non-pathogenic mycobacterial model organism Mycobacterium smegmatis (Msmeg) was used to reveal the structure and function of MSMEG_3955; which is a homologue of Rv3131 from Mtb. Using chromatography and spectroscopy techniques it was revealed that cofactor flavin mononucleotide (FMN) was bound to flavoprotein MSMEG_3955. Consistent with the homology modelling predictions, Circular Dichroism (CD) analysis indicated that the MSMEG_3955 is composed of 39.3% α-helix and 24.9% ß-pleated sheets. In contrast to the current notions, the enzymatic assays performed in the present study revealed that MSMEG_3955 was not capable of reducing nitro substrates but showed NADPH dependent FMN oxidoreductase activity. Also, gel permeation chromatography, dynamic light scattering and native acidic gels showed that MSMEG_3955 exists as a homotrimer. Furthermore, the presence of NADPH dependent FMN oxidoreductase and homotrimeric existence could be an alternative function of the protein to help the bacteria survive in dormant state or may be involved in other biochemical pathways. CONCLUSION: MSMEG_3955 is a FMN bound flavoprotein, which exits as a trimer under in vitro conditions. There is no disulphide linkages in between the three protomers of the homotrimer MSMEG_3955. It has a NADPH dependent FMN oxidoreductase activity.


Assuntos
Proteínas de Bactérias/metabolismo , FMN Redutase/metabolismo , Mycobacterium smegmatis/enzimologia , NADH NADPH Oxirredutases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Dimerização , FMN Redutase/química , FMN Redutase/genética , Mononucleotídeo de Flavina/metabolismo , Mycobacterium smegmatis/química , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , NAD/metabolismo , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , NADP/metabolismo
11.
Mol Microbiol ; 116(5): 1361-1377, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34614242

RESUMO

This study identifies a post-transcriptional mechanism of iron uptake regulation by Puf2 and Puf4 of the Pumilio and FBF (Puf) family of RNA-binding proteins in Schizosaccharomyces pombe. Cells expressing Puf2 and Puf4 stimulate decay of the frp1+ mRNA encoding a key enzyme of the reductive iron uptake pathway. Results consistently showed that frp1+ mRNA is stabilized in puf2Δ puf4Δ mutant cells under iron-replete conditions. As a result, puf2Δ puf4Δ cells exhibit an increased sensitivity to iron accompanied by enhanced ferrireductase activity. A pool of GFP-frp1+ 3'UTR RNAs was generated using a reporter gene containing the 3' untranslated region (UTR) of frp1+ that was under the control of a regulatable promoter. Results showed that Puf2 and Puf4 accelerate the destabilization of mRNAs containing the frp1+ 3'UTR which harbors two Pumilio response elements (PREs). Binding studies revealed that the PUM-homology RNA-binding domain of Puf2 and Puf4 expressed in Escherichia coli specifically interacts with PREs in the frp1+ 3'UTR. Using RNA immunoprecipitation in combination with reverse transcription qPCR assays, results showed that Puf2 and Puf4 interact preferentially with frp1+ mRNA under basal and iron-replete conditions, thereby contributing to inhibit Frp1 production and protecting cells against toxic levels of iron.


Assuntos
FMN Redutase/genética , FMN Redutase/metabolismo , Ferro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Regiões 3' não Traduzidas , DNA Fúngico , Regulação Fúngica da Expressão Gênica , Mutação , Regiões Promotoras Genéticas , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
12.
Blood ; 138(22): 2216-2230, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34232987

RESUMO

Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma, the leading childhood cancer in sub-Saharan Africa. Burkitt cells retain aspects of germinal center B-cell physiology with MYC-driven B-cell hyperproliferation; however, little is presently known about their iron metabolism. CRISPR/Cas9 analysis highlighted the little-studied ferrireductase CYB561A3 as critical for Burkitt proliferation but not for that of the closely related EBV-transformed lymphoblastoid cells or nearly all other Cancer Dependency Map cell lines. Burkitt CYB561A3 knockout induced profound iron starvation, despite ferritinophagy ad plasma membrane transferrin upregulation. Elevated concentrations of ascorbic acid, a key CYB561 family electron donor, or the labile iron source ferrous citrate rescued Burkitt CYB561A3 deficiency. CYB561A3 knockout caused catastrophic lysosomal and mitochondrial damage and impaired mitochondrial respiration. Conversely, lymphoblastoid B cells with the transforming EBV latency III program were instead dependent on the STEAP3 ferrireductase. These results highlight CYB561A3 as an attractive therapeutic Burkitt lymphoma target.


Assuntos
Linfoma de Burkitt/patologia , Citocromos b/genética , Regulação Neoplásica da Expressão Gênica , Lisossomos/patologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Linfoma de Burkitt/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células , Infecções por Vírus Epstein-Barr/complicações , FMN Redutase/genética , Células HEK293 , Herpesvirus Humano 4/isolamento & purificação , Humanos , Lisossomos/genética , Mitocôndrias/genética , Mitocôndrias/patologia
13.
FEBS Open Bio ; 11(7): 1981-1986, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34043290

RESUMO

In this study, we investigated the stereospecificity of hydride transfer from NADH to flavin mononucleotide (FMN) in reactions catalyzed by the FMN-dependent NADH-indigo reductase expressed by thermophilic Bacillus smithii. We performed 1 H-NMR spectroscopy using deuterium-labeled NADH (4R-2 H-NADH) and molecular docking simulations to reveal that the pro-S hydrogen at the C4 position of the nicotinamide moiety in NADH was specifically transferred to the flavin-N5 atom of FNM. Altogether, our findings may aid in the improvement of the indigo dyeing (Aizome) process.


Assuntos
FMN Redutase , Mononucleotídeo de Flavina , Bacillus , Mononucleotídeo de Flavina/química , Índigo Carmim , Simulação de Acoplamento Molecular , NAD/química
14.
Arch Biochem Biophys ; 704: 108874, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33862020

RESUMO

The short-chain flavin reductases BorF and AbeF reduce FAD to FADH2, which is then used by flavin-dependent halogenases (BorH and AbeH respectively) to regioselectively chlorinate tryptophan in the biosynthesis of indolotryptoline natural products. Recombinant AbeF and BorF were overexpressed and purified as homodimers from E. coli, and copurified with substoichiometric amounts of FAD, which could be easily removed. AbeF and BorF can reduce FAD, FMN, and riboflavin in vitro and are selective for NADH over NADPH. Initial velocity studies in the presence and absence of inhibitors showed that BorF proceeds by a sequential ordered kinetic mechanism in which FAD binds first, while AbeF follows a random-ordered sequence of substrate binding. Fluorescence quenching experiments verified that NADH does not bind BorF in the absence of FAD, and that both AbeF and BorF bind FAD with higher affinity than FADH2. pH-rate profiles of BorF and AbeF were bell-shaped with maximum kcat at pH 7.5, and site-directed mutagenesis of BorF implicated His160 and Arg38 as contributing to the catalytic activity and the pH dependence.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , FMN Redutase/química , Mononucleotídeo de Flavina/química , Flavina-Adenina Dinucleotídeo/química , Riboflavina/química , Cinética
15.
J Inorg Biochem ; 218: 111407, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33684686

RESUMO

Iron is a necessary element for nearly all forms of life, and the ability to acquire this trace nutrient has been identified as a key virulence factor for the establishment of infection by unicellular pathogens. In the presence of O2, iron typically exists in the ferric (Fe3+) oxidation state, which is highly unstable in aqueous conditions, necessitating its sequestration into cofactors and/or host proteins to remain soluble. To counter this insolubility, and to compete with host sequestration mechanisms, many unicellular pathogens will secrete low molecular weight, high-affinity Fe3+ chelators known as siderophores. Once acquired, unicellular pathogens must liberate the siderophore-bound Fe3+ in order to assimilate this nutrient into metabolic pathways. While these organisms may hydrolyze the siderophore backbone to release the chelated Fe3+, this approach is energetically costly. Instead, iron may be liberated from the Fe3+-siderophore complex through reduction to Fe2+, which produces a lower-affinity form of iron that is highly soluble. This reduction is performed by a class of enzymes known as ferric reductases. Ferric reductases are broadly-distributed electron-transport proteins that are expressed by numerous infectious organisms and are connected to the virulence of unicellular pathogens. Despite this importance, ferric reductases remain poorly understood. This review provides an overview of our current understanding of unicellular ferric reductases (both soluble and membrane-bound), with an emphasis on the important but underappreciated connection between ferric-reductase mediated Fe3+ reduction and the transport of Fe2+ via ferrous iron transporters.


Assuntos
Eucariotos/metabolismo , FMN Redutase/metabolismo , Compostos Ferrosos/metabolismo , Transporte Biológico , Homeostase , Oxirredução
16.
J Biol Inorg Chem ; 26(2-3): 313-326, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33559753

RESUMO

Iron is a fundamental element for virtually all forms of life. Despite its abundance, its bioavailability is limited, and thus, microbes developed siderophores, small molecules, which are synthesized inside the cell and then released outside for iron scavenging. Once inside the cell, iron removal does not occur spontaneously, instead this process is mediated by siderophore-interacting proteins (SIP) and/or by ferric-siderophore reductases (FSR). In the past two decades, representatives of the SIP subfamily have been structurally and biochemically characterized; however, the same was not achieved for the FSR subfamily. Here, we initiate the structural and functional characterization of FhuF, the first and only FSR ever isolated. FhuF is a globular monomeric protein mainly composed by α-helices sheltering internal cavities in a fold resembling the "palm" domain found in siderophore biosynthetic enzymes. Paramagnetic NMR spectroscopy revealed that the core of the cluster has electronic properties in line with those of previously characterized 2Fe-2S ferredoxins and differences appear to be confined to the coordination of Fe(III) in the reduced protein. In particular, the two cysteines coordinating this iron appear to have substantially different bond strengths. In similarity with the proteins from the SIP subfamily, FhuF binds both the iron-loaded and the apo forms of ferrichrome in the micromolar range and cyclic voltammetry reveals the presence of redox-Bohr effect, which broadens the range of ferric-siderophore substrates that can be thermodynamically accessible for reduction. This study suggests that despite the structural differences between FSR and SIP proteins, mechanistic similarities exist between the two classes of proteins.


Assuntos
Escherichia coli/enzimologia , FMN Redutase/química , FMN Redutase/metabolismo , Cisteína/metabolismo , Modelos Moleculares , Oxirredução , Domínios Proteicos
17.
Biomolecules ; 11(1)2021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33451048

RESUMO

Among seven homologs of cytochrome b561 in a model organism C. elegans, Cecytb-2 was confirmed to be expressed in digestive organs and was considered as a homolog of human Dcytb functioning as a ferric reductase. Cecytb-2 protein was expressed in Pichia pastoris cells, purified, and reconstituted into a phospholipid bilayer nanodisc. The reconstituted Cecytb-2 in nanodisc environments was extremely stable and more reducible with ascorbate than in a detergent-micelle state. We confirmed the ferric reductase activity of Cecytb-2 by analyzing the oxidation of ferrous heme upon addition of ferric substrate under anaerobic conditions, where clear and saturable dependencies on the substrate concentrations following the Michaelis-Menten equation were observed. Further, we confirmed that the ferric substrate was converted to a ferrous state by using a nitroso-PSAP assay. Importantly, we observed that the ferric reductase activity of Cecytb-2 became enhanced in the phospholipid bilayer nanodisc.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , FMN Redutase/metabolismo , L-Lactato Desidrogenase (Citocromo)/metabolismo , Bicamadas Lipídicas/metabolismo , Nanopartículas/química , Fosfolipídeos/metabolismo , Animais , Proteínas de Caenorhabditis elegans/isolamento & purificação , Detergentes/farmacologia , Difusão Dinâmica da Luz , Glucosídeos/farmacologia , L-Lactato Desidrogenase (Citocromo)/isolamento & purificação , Micelas , Tamanho da Partícula , Bases de Schiff
18.
Biochemistry ; 60(1): 31-40, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33350810

RESUMO

The flavin reductase (FRED) and isobutylamine N-hydroxylase (IBAH) from Streptomyces viridifaciens constitute a two-component, flavin-dependent monooxygenase system that catalyzes the first step in valanimycin biosynthesis. FRED is an oxidoreductase that provides the reduced flavin to IBAH, which then catalyzes the hydroxylation of isobutylamine (IBA) to isobutylhydroxylamine (IBHA). In this work, we used several complementary methods to investigate FAD binding, steady-state and rapid reaction kinetics, and enzyme-enzyme interactions in the FRED:IBAH system. The affinity of FRED for FADox is higher than its affinity for FADred, consistent with its function as a flavin reductase. Conversely, IBAH binds FADred more tightly than FADox, consistent with its role as a monooxygenase. FRED exhibits a strong preference (28-fold) for NADPH over NADH as the electron source for FAD reduction. Isothermal titration calorimetry was used to study the association of FRED and IBAH. In the presence of FAD, either oxidized or reduced, FRED and IBAH associate with a dissociation constant of 7-8 µM. No interaction was observed in the absence of FAD. These results are consistent with the formation of a protein-protein complex for direct transfer of reduced flavin from the reductase to the monooxygenase in this two-component system.


Assuntos
Proteínas de Bactérias/metabolismo , FMN Redutase/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Oxigenases de Função Mista/metabolismo , Streptomyces/enzimologia , Compostos Azo/metabolismo , Hidroxilação , Cinética , NADPH Oxidases/metabolismo , Consumo de Oxigênio
19.
Nat Chem Biol ; 17(1): 104-112, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33139950

RESUMO

Tyrian purple, mainly composed of 6,6'-dibromoindigo (6BrIG), is an ancient dye extracted from sea snails and was recently demonstrated as a biocompatible semiconductor material. However, its synthesis remains limited due to uncharacterized biosynthetic pathways and the difficulty of regiospecific bromination. Here, we introduce an effective 6BrIG production strategy in Escherichia coli using tryptophan 6-halogenase SttH, tryptophanase TnaA and flavin-containing monooxygenase MaFMO. Since tryptophan halogenases are expressed in highly insoluble forms in E. coli, a flavin reductase (Fre) that regenerates FADH2 for the halogenase reaction was used as an N-terminal soluble tag of SttH. A consecutive two-cell reaction system was designed to overproduce regiospecifically brominated precursors of 6BrIG by spatiotemporal separation of bromination and bromotryptophan degradation. These approaches led to 315.0 mg l-1 6BrIG production from tryptophan and successful synthesis of regiospecifically dihalogenated indigos. Furthermore, it was demonstrated that 6BrIG overproducing cells can be directly used as a bacterial dye.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , FMN Redutase/genética , Regulação Bacteriana da Expressão Gênica , Indóis/metabolismo , Oxirredutases/genética , Oxigenases/genética , Triptofano/metabolismo , Triptofanase/genética , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Clonagem Molecular , Corantes/isolamento & purificação , Corantes/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , FMN Redutase/metabolismo , Flavina-Adenina Dinucleotídeo/análogos & derivados , Flavina-Adenina Dinucleotídeo/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Halogenação , Índigo Carmim/isolamento & purificação , Índigo Carmim/metabolismo , Indóis/isolamento & purificação , Engenharia Metabólica/métodos , Oxirredutases/metabolismo , Oxigenases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Semicondutores , Estereoisomerismo , Triptofanase/metabolismo
20.
Int J Mol Sci ; 22(1)2020 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-33374479

RESUMO

Sorbus commixta is a valuable hardwood plant with a high economical value for its medicinal and ornamental qualities. The aim of this work was to investigate the effects of the iron (Fe) source and medium pH on the growth and development of S. commixta in vitro. The Fe sources used, including non-chelated iron sulfate (FeSO4), iron ethylenediaminetetraacetic acid (Fe-EDTA), and iron diethylenetriaminepentaacetic acid (Fe-DTPA), were supplemented to the Multipurpose medium with a final Fe concentration of 2.78 mg·L-1. The medium without any supplementary Fe was used as the control. The pH of the agar-solidified medium was adjusted to either 4.70, 5.70, or 6.70. The experiment was conducted in a culture room for six weeks with 25 °C day and night temperatures, and a 16-h photoperiod with a light intensity of 50 mmol·m-2·s-1 photosynthetic photon flux density (PPFD). Both the Fe source and pH affected the growth and development of the micropropagated plants in vitro. The leaves were greener in the pH 4.70 and 5.70 treatments. The tissue Fe content decreased with the increase of the medium pH. The leaf chlorophyll content was similar between plants treated with FeSO4 and those with Fe-EDTA. The numbers of the shoots and roots of plantlets treated with FeSO4 were 2.5 and 2 times greater than those of the control, respectively. The fresh and dry weights of the shoot and the root were the greatest for plants treated with Fe-EDTA combined with pH 5.70. The calcium, magnesium, and manganese contents in the plantlets increased in the pH 5.70 treatments regardless of the Fe source. Supplementary Fe decreased the activity of ferric chelate reductase. Overall, although the plantlets absorbed more Fe at pH 4.70, Fe-EDTA combined with pH 5.70 was found to be the best for the growth and development of S. commixta in vitro.


Assuntos
Meios de Cultura/farmacologia , Compostos Férricos/química , Compostos Ferrosos/química , Ácido Pentético/análogos & derivados , Sorbus/crescimento & desenvolvimento , Antioxidantes/química , Clorofila/química , Ácido Edético/química , FMN Redutase/química , Concentração de Íons de Hidrogênio , Ferro , Ácido Pentético/química , Fotossíntese , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Estômatos de Plantas/metabolismo , Sorbus/metabolismo , Fatores de Tempo
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