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1.
Phys Chem Chem Phys ; 23(37): 20867-20874, 2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34374395

RESUMO

Cyanobacteriochromes (CBCRs) are bi-stable photoreceptor proteins with high potential for biotechnological applications. Most of these proteins utilize phycocyanobilin (PCB) as a light-sensing co-factor, which is unique to cyanobacteria, but some variants also incorporate biliverdin (BV). The latter are of particular interest for biotechnology due to the natural abundance and red-shifted absorption of BV. Here, AmI-g2 was investigated, a CBCR capable of binding both PCB and BV. The assembly kinetics and primary photochemistry of AmI-g2 with both chromophores were studied in vitro. The assembly reaction with PCB is roughly 10× faster than BV, and the formation of a non-covalent intermediate was identified as the rate-limiting step in the case of BV. This step is fast for PCB, where the formation of the covalent thioether bond between AmI-g2 and PCB becomes rate-limiting. The photochemical quantum yields of the forward and backward reactions of AmI-g2 were estimated and discussed in the context of homologous CBCRs.


Assuntos
Biliverdina/química , Cianobactérias/metabolismo , Fotorreceptores Microbianos/química , Ficobilinas/química , Ficocianina/química , Biliverdina/metabolismo , Cinética , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo , Ficobilinas/metabolismo , Ficocianina/metabolismo , Ligação Proteica , Teoria Quântica , Espectrofotometria
3.
Sci Adv ; 7(22)2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33888467

RESUMO

The coronaviral spike is the dominant viral antigen and the target of neutralizing antibodies. We show that SARS-CoV-2 spike binds biliverdin and bilirubin, the tetrapyrrole products of heme metabolism, with nanomolar affinity. Using cryo-electron microscopy and x-ray crystallography, we mapped the tetrapyrrole interaction pocket to a deep cleft on the spike N-terminal domain (NTD). At physiological concentrations, biliverdin significantly dampened the reactivity of SARS-CoV-2 spike with immune sera and inhibited a subset of neutralizing antibodies. Access to the tetrapyrrole-sensitive epitope is gated by a flexible loop on the distal face of the NTD. Accompanied by profound conformational changes in the NTD, antibody binding requires relocation of the gating loop, which folds into the cleft vacated by the metabolite. Our results indicate that SARS-CoV-2 spike NTD harbors a dominant epitope, access to which can be controlled by an allosteric mechanism that is regulated through recruitment of a metabolite.


Assuntos
COVID-19/imunologia , Heme/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/imunologia , Bilirrubina/metabolismo , Biliverdina/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Epitopos , Humanos , Soros Imunes , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade
4.
J Biol Chem ; 296: 100217, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33839679

RESUMO

Heme oxygenase (HO) converts heme to carbon monoxide, biliverdin, and free iron, products that are essential in cellular redox signaling and iron recycling. In higher plants, HO is also involved in the biosynthesis of photoreceptor pigment precursors. Despite many common enzymatic reactions, the amino acid sequence identity between plant-type and other HOs is exceptionally low (∼19.5%), and amino acids that are catalytically important in mammalian HO are not conserved in plant-type HOs. Structural characterization of plant-type HO is limited to spectroscopic characterization by electron spin resonance, and it remains unclear how the structure of plant-type HO differs from that of other HOs. Here, we have solved the crystal structure of Glycine max (soybean) HO-1 (GmHO-1) at a resolution of 1.06 Å and carried out the isothermal titration calorimetry measurements and NMR spectroscopic studies of its interaction with ferredoxin, the plant-specific electron donor. The high-resolution X-ray structure of GmHO-1 reveals several novel structural components: an additional irregularly structured region, a new water tunnel from the active site to the surface, and a hydrogen-bonding network unique to plant-type HOs. Structurally important features in other HOs, such as His ligation to the bound heme, are conserved in GmHO-1. Based on combined data from X-ray crystallography, isothermal titration calorimetry, and NMR measurements, we propose the evolutionary fine-tuning of plant-type HOs for ferredoxin dependency in order to allow adaptation to dynamic pH changes on the stroma side of the thylakoid membrane in chloroplast without losing enzymatic activity under conditions of fluctuating light.


Assuntos
Ferredoxinas/química , Heme Oxigenase-1/química , Heme/química , Ferro/química , Proteínas de Plantas/química , Soja/química , Sequência de Aminoácidos , Biliverdina/química , Biliverdina/metabolismo , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Domínio Catalítico , Cloroplastos/química , Cloroplastos/enzimologia , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Ferredoxinas/genética , Ferredoxinas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Heme/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Ligação de Hidrogênio , Ferro/metabolismo , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Soja/enzimologia , Soja/genética , Tilacoides/química , Tilacoides/enzimologia
5.
J Biol Chem ; 296: 100666, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33862082

RESUMO

Heme oxygenases (HOs) play a critical role in recouping iron from the labile heme pool. The acquisition and liberation of heme iron are especially important for the survival of pathogenic bacteria. All characterized HOs, including those belonging to the HugZ superfamily, preferentially cleave free b-type heme. Another common form of heme found in nature is c-type heme, which is covalently linked to proteinaceous cysteine residues. However, mechanisms for direct iron acquisition from the c-type heme pool are unknown. Here we identify a HugZ homolog from the oligopeptide permease (opp) gene cluster of Paracoccus denitrificans that lacks any observable reactivity with heme b and show that it instead rapidly degrades c-type hemopeptides. This c-type heme oxygenase catalyzes the oxidative cleavage of the model substrate microperoxidase-11 at the ß- and/or δ-meso position(s), yielding the corresponding peptide-linked biliverdin, CO, and free iron. X-ray crystallographic analysis suggests that the switch in substrate specificity from b-to c-type heme involves loss of the N-terminal α/ß domain and C-terminal loop containing the coordinating histidine residue characteristic of HugZ homologs, thereby accommodating a larger substrate that provides its own iron ligand. These structural features are also absent in certain heme utilization/storage proteins from human pathogens that exhibit low or no HO activity with free heme. This study thus expands the scope of known iron acquisition strategies to include direct oxidative cleavage of heme-containing proteolytic fragments of c-type cytochromes and helps to explain why certain oligopeptide permeases show specificity for the import of heme in addition to peptides.


Assuntos
Proteínas de Bactérias/metabolismo , Biliverdina/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Heme/análogos & derivados , Heme/metabolismo , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Paracoccus denitrificans/enzimologia , Catálise , Cristalografia por Raios X , Heme Oxigenase (Desciclizante)/química , Especificidade por Substrato
6.
J Chem Phys ; 154(6): 065101, 2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33588533

RESUMO

An enhanced interest in the phytochrome-based fluorescent proteins is explained by their ability to absorb and emit light in the far-red and infra-red regions particularly suitable for bioimaging. The fluorescent protein IFP1.4 was engineered from the chromophore-binding domain of a bacteriophytochrome in attempts to increase the fluorescence quantum yield. We report the results of simulations of structures in the ground S0 and excited S1 electronic states of IFP1.4 using the methods of quantum chemistry and quantum mechanics/molecular mechanics. We construct different protonation states of the biliverdin (BV) chromophore in the red-absorbing form of the protein by moving protons from the BV pyrrole rings to a suitable acceptor within the system and show that these structures are close in energy but differ by absorption bands. For the first time, we report structures of the minimum energy conical intersection points S1/S0 on the energy surfaces of BV in the protein environment and describe their connection to the local minima in the excited S1 state. These simulations allow us to characterize the deactivation routes in IFP1.4.


Assuntos
Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Simulação de Dinâmica Molecular , Fitocromo/metabolismo , Proteínas de Bactérias , Biliverdina/metabolismo , Ligação de Hidrogênio , Conformação Proteica , Domínios Proteicos , Teoria Quântica
7.
Eur J Pharm Sci ; 159: 105684, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33359481

RESUMO

BACKGROUND AND PURPOSE: Biliverdin (BV) administration induces antioxidant and anti-inflammatory effects, with previous reports also identifying anti-anaphylactic potential. Interestingly however, intra-duodenal administration of BV in rats leads to the formation of bilirubin-10-sulfonate (BRS), which might be responsible for BV's purported effects. EXPERIMENTAL APPROACH: This study aimed to assess the intravenous, intraperitoneal and intraduodenal pharmacokinetics of BRS and BV in order to assess their therapeutic potential in future studies. Bile and venous blood were intermittently collected before and after administration, which was subsequently analysed using liquid chromatography-mass spectrometry for quantification of bile pigment concentrations. KEY RESULTS: Interestingly, i.p. BRS administration led to a greater circulating concentration and had a reduced excretion rate, which resulted in a substantially elevated AUC180 when compared to BV administration. Furthermore, BRS was excreted intact in the bile, in contrast to BV which was excreted after chemical reduction and conjugation. Intraperitoneal and intraduodenal administration substantially increased blood BRS concentrations (p<0.05), however, the bioavailability of BV was higher than BRS following i.p. administration (i.p. BV 28.4%, BRS 15.5%) but lower following i.d. administration (i.d. BV 0.04%, BRS 0.07%), over 180 minutes. When BRS was administered i.v., BRS had a significantly (p<0.05) longer distribution (191.9 vs 54.1 minutes) half-life compared to BV, and significantly reduced (p<0.05) volume of distribution (0.026 vs 0.145 L kg-1). As a consequence, intraperitoneal and intraduodenal administration resulted in significantly greater blood concentrations of BRS (p<0.05) over 180 minutes. Therefore, BRS may be more likely to induce antioxidant or molecular effects, when compared to BV, due to greater concentrations and a longer half-life. CONCLUSIONS AND IMPLICATIONS: Cumulatively, these data demonstrate that BRS has a superior pharmacokinetic profile when compared to BV, which is a result of its resistance to hepatic metabolism and excretion. These data therefore provide a basis to explore the capacity of BRS to protect from inflammatory pathology.


Assuntos
Bilirrubina , Biliverdina , Animais , Antioxidantes , Bile/metabolismo , Biliverdina/metabolismo , Disponibilidade Biológica , Ratos
8.
Int J Mol Sci ; 21(17)2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32872628

RESUMO

Cyanobacteriochromes (CBCRs), which are known as linear tetrapyrrole-binding photoreceptors, to date can only be detected from cyanobacteria. They can perceive light only in a small unit, which is categorized into various lineages in correlation with their spectral and structural characteristics. Recently, we have succeeded in identifying specific molecules, which can incorporate mammalian intrinsic biliverdin (BV), from the expanded red/green (XRG) CBCR lineage and in converting BV-rejective molecules into BV-acceptable ones with the elucidation of the structural basis. Among the BV-acceptable molecules, AM1_1870g3_BV4 shows a spectral red-shift in comparison with other molecules, while NpF2164g5_BV4 does not show photoconversion but stably shows a near-infrared (NIR) fluorescence. In this study, we found that AM1_1870g3_BV4 had a specific Tyr residue near the d-ring of the chromophore, while others had a highly conserved Leu residue. The replacement of this Tyr residue with Leu in AM1_1870g3_BV4 resulted in a blue-shift of absorption peak. In contrast, reverse replacement in NpF2164g5_BV4 resulted in a red-shift of absorption and fluorescence peaks, which applies to fluorescence bio-imaging in mammalian cells. Notably, the same Tyr/Leu-dependent color-tuning is also observed for the CBCRs belonging to the other lineage, which indicates common molecular mechanisms.


Assuntos
Proteínas de Bactérias/metabolismo , Biliverdina/metabolismo , Cianobactérias/metabolismo , Fotorreceptores Microbianos/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Biliverdina/química , Cor , Células HeLa , Humanos , Luz , Homologia de Sequência
9.
Proc Natl Acad Sci U S A ; 117(31): 18574-18581, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32661155

RESUMO

Many vertebrates have distinctive blue-green bones and other tissues due to unusually high biliverdin concentrations-a phenomenon called chlorosis. Despite its prevalence, the biochemical basis, biology, and evolution of chlorosis are poorly understood. In this study, we show that the occurrence of high biliverdin in anurans (frogs and toads) has evolved multiple times during their evolutionary history, and relies on the same mechanism-the presence of a class of serpin family proteins that bind biliverdin. Using a diverse combination of techniques, we purified these serpins from several species of nonmodel treefrogs and developed a pipeline that allowed us to assemble their complete amino acid and nucleotide sequences. The described proteins, hereafter named biliverdin-binding serpins (BBS), have absorption spectra that mimic those of phytochromes and bacteriophytochromes. Our models showed that physiological concentration of BBSs fine-tune the color of the animals, providing the physiological basis for crypsis in green foliage even under near-infrared light. Additionally, we found that these BBSs are most similar to human glycoprotein alpha-1-antitrypsin, but with a remarkable functional diversification. Our results present molecular and functional evidence of recurrent evolution of chlorosis, describe a biliverdin-binding protein in vertebrates, and introduce a function for a member of the serpin superfamily, the largest and most ubiquitous group of protease inhibitors.


Assuntos
Anuros/fisiologia , Biliverdina/metabolismo , Serpinas/metabolismo , Pigmentação da Pele/fisiologia , Animais , Anuros/classificação , Anuros/genética , Biliverdina/química , Mimetismo Biológico/fisiologia , Serpinas/química , Serpinas/genética , Pigmentação da Pele/genética
10.
J Exp Biol ; 223(Pt 6)2020 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-32054680

RESUMO

Infection can result in substantial costs to animals, so they frequently respond by removing infectious agents with an immune response. However, immune responses entail their own costs, including upregulation of processes that destroy pathogens (e.g. the production of reactive oxygen species) and processes that limit the extent of self-damage during the immune response (e.g. production of anti-inflammatory proteins such as haptoglobin). Here, we simulated bacterial infection across a 1000-fold range using lipopolysaccharide (LPS) administered to northern bobwhite quail (Colinus virginianus), and quantified metrics related to pro-inflammatory conditions [i.e. generation of oxidative damage (d-ROMs), depletion of antioxidant capacity], anti-inflammatory mechanisms (i.e. production of haptoglobin, expression of the enzyme heme oxygenase, production of the organic molecule biliverdin) and nutritional physiology (e.g. circulating triglyceride levels, maintenance of body mass). We detected increases in levels of haptoglobin and d-ROMs even at LPS doses that are 1/1000th the concentration of doses frequently used in ecoimmunological studies, while loss of body mass and decreases in circulating triglycerides manifested only in individuals receiving the highest dose of LPS (1 mg LPS kg-1 body mass), highlighting variation among dose-dependent responses. Additionally, individuals that lost body mass during the course of the experiment had lower levels of circulating triglycerides, and those with more oxidative damage had greater levels of heme oxygenase expression, which highlights the complex interplay between pro- and anti-inflammatory processes. Because low doses of LPS may simulate natural infection levels, variation in dose-dependent physiological responses may be particularly important in modeling how free-living animals navigate immune challenges.


Assuntos
Colinus , Estresse Oxidativo , Animais , Biliverdina/metabolismo , Heme Oxigenase-1 , Lipopolissacarídeos/toxicidade , Espécies Reativas de Oxigênio
11.
Front Immunol ; 11: 66, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32082323

RESUMO

Heme is one of the most abundant molecules in the body acting as the functional core of hemoglobin/myoglobin involved in the O2/CO2 carrying in the blood and tissues, redox enzymes and cytochromes in mitochondria. However, free heme is toxic and therefore its removal is a significant priority for the host. Heme is a well-established danger-associated molecular pattern (DAMP), which binds to toll-like receptor 4 (TLR4) to induce immune responses. Heme-derived metabolites including the bile pigments, biliverdin (BV) and bilirubin (BR), were first identified as toxic drivers of neonatal jaundice in 1800 but have only recently been appreciated as endogenous drivers of multiple signaling pathways involved in protection from oxidative stress and regulators of immune responses. The tissue concentration of heme, BV and BR is tightly controlled. Heme oxygenase-1 (HO-1, encoded by HMOX1) produces BV by heme degradation, while biliverdin reductase-A (BLVR-A) generates BR by the subsequent conversion of BV. BLVR-A is a fascinating protein that possesses a classical protein kinase domain, which is activated in response to BV binding to its enzymatic site and initiates the downstream mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. This links BLVR-A activity to cell growth and survival pathways. BLVR-A also contains a bZip DNA binding domain and a nuclear export sequence (NES) and acts as a transcription factor to regulate the expression of immune modulatory genes. Here we will discuss the role of heme-related immune response and the potential for targeting the heme system for therapies directed toward hepatitis and cancer.


Assuntos
Heme/imunologia , Heme/metabolismo , Imunidade/fisiologia , Inflamação/imunologia , Animais , Bilirrubina/imunologia , Bilirrubina/metabolismo , Biliverdina/imunologia , Biliverdina/metabolismo , Humanos , Inflamação/metabolismo
12.
Biochemistry ; 59(9): 1023-1037, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32073262

RESUMO

Phytochromes are biological photoswitches that interconvert between two parent states (Pr and Pfr). The transformation is initiated by photoisomerization of the tetrapyrrole chromophore, followed by a sequence of chromophore and protein structural changes. In the last step, a phytochrome-specific peptide segment (tongue) undergoes a secondary structure change, which in prokaryotic phytochromes is associated with the (de)activation of the output module. The focus of this work is the Pfr-to-Pr photoconversion of the bathy bacteriophytochrome Agp2 in which Pfr is the thermodynamically stable state. Using spectroscopic techniques, we studied the structural and functional consequences of substituting Arg211, Tyr165, His278, and Phe192 close to the biliverdin (BV) chromophore. In Pfr, substitutions of these residues do not affect the BV structure. The characteristic Pfr properties of bathy phytochromes, including the protonated propionic side chain of ring C (propC) of BV, are preserved. However, replacing Arg211 or Tyr165 blocks the photoconversion in the Meta-F state, prior to the secondary structure transition of the tongue and without deprotonation of propC. The Meta-F state of these variants displays low photochemical activity, but electronic excitation causes ultrafast alterations of the hydrogen bond network surrounding the chromophore. In all variants studied here, thermal back conversion from the photoproducts to Pfr is decelerated but substitution of His278 or Phe192 is not critical for the Pfr-to-Pr photoconversion. These variants do not impair deprotonation of propC or the α-helix/ß-sheet transformation of the tongue during the Meta-F-to-Pr decay. Thus, we conclude that propC deprotonation is essential for restructuring of the tongue.


Assuntos
Biliverdina/metabolismo , Fitocromo/química , Fitocromo/ultraestrutura , Agrobacterium tumefaciens , Proteínas de Bactérias/química , Ligação de Hidrogênio , Luz , Fitocromo/fisiologia , Prótons , Análise Espectral Raman/métodos , Tetrapirróis/química , Tetrapirróis/metabolismo
13.
Biochem J ; 477(3): 601-614, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31913441

RESUMO

The pro-oxidant effect of free heme (Fe2+-protoporphyrin IX) is neutralized by phylogenetically-conserved heme oxygenases (HMOX) that generate carbon monoxide, free ferrous iron, and biliverdin (BV) tetrapyrrole(s), with downstream BV reduction by non-redundant NADPH-dependent BV reductases (BLVRA and BLVRB) that retain isomer-restricted functional activity for bilirubin (BR) generation. Regioselectivity for the heme α-meso carbon resulting in predominant BV IXα generation is a defining characteristic of canonical HMOXs, thereby limiting generation and availability of BVs IXß, IXδ, and IXγ as BLVRB substrates. We have now exploited the unique capacity of the Pseudomonas aeruginosa (P. aeruginosa) hemO/pigA gene for focused generation of isomeric BVs (IXß and IXδ). A scalable system followed by isomeric separation yielded highly pure samples with predicted hydrogen-bonded structure(s) as documented by 1H NMR spectroscopy. Detailed kinetic studies established near-identical activity of BV IXß and BV IXδ as BLVRB-selective substrates, with confirmation of an ordered sequential mechanism of BR/NADP+ dissociation. Halogenated xanthene-based compounds previously identified as BLVRB-targeted flavin reductase inhibitors displayed comparable inhibition parameters using BV IXß as substrate, documenting common structural features of the cofactor/substrate-binding pocket. These data provide further insights into structure/activity mechanisms of isomeric BVs as BLVRB substrates, with potential applicability to further dissect redox-regulated functions in cytoprotection and hematopoiesis.


Assuntos
Biliverdina , Heme Oxigenase (Desciclizante) , Heme/metabolismo , Pseudomonas aeruginosa/metabolismo , Biliverdina/química , Biliverdina/metabolismo , Genes Bacterianos/fisiologia , Heme Oxigenase (Desciclizante)/química , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Cinética , Oxirredução , Oxirredutases/metabolismo , Pseudomonas aeruginosa/genética
14.
Artigo em Inglês | MEDLINE | ID: mdl-31704186

RESUMO

When exposed to stressors, animals physiologically respond by secreting glucocorticoid hormones. Most birds, reptiles, and amphibians secrete corticosterone (CORT), which allows them to maximize short-term survival, including by modulating lipid metabolism. However, the factors regulating lipid metabolism, particularly during acute (i.e., short-term) stressors, are not well-characterized. To investigate one putative mechanism, we examined how expression of the enzyme heme oxygenase (HO), which primarily converts heme into biliverdin, changes during an acute stressor. Because HO has links to decreased levels of triglycerides, we tested the hypothesis that an acute stressor increases HO expression, which would concomitantly decrease circulating lipid levels. We compared free-living house sparrow (Passer domesticus) nestlings exposed to a one-hour stressor to control individuals, and quantified HO expression and biliverdin concentration in spleen, liver, or kidney tissue, as well as circulating CORT, triglyceride, and glycerol levels. Nestlings exposed to a stressor had reduced circulating triglycerides consistent with an increased rate of gluconeogenesis during an acute stressor. Concentrations of triglycerides were also negatively correlated with HO expression in the liver, which is consistent with mammalian studies. However, contrary to our predictions, exposure to a stressor did not affect HO expression, or biliverdin concentration in liver, spleen, or kidney. Overall, our results support links between CORT, triglyceride levels, and HO expression, though the molecular pathways connecting these metrics still need to be elucidated.


Assuntos
Biliverdina/metabolismo , Corticosterona/sangue , Heme Oxigenase (Desciclizante)/metabolismo , Pardais/metabolismo , Estresse Fisiológico , Triglicerídeos/sangue , Animais , Rim/metabolismo , Fígado/metabolismo , Baço/metabolismo
15.
FEBS J ; 287(2): 284-294, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31319014

RESUMO

Substrate channeling is a widespread mechanism in metabolic pathways to avoid decomposition of unstable intermediates, competing reactions, and to accelerate catalytic turnover. During the biosynthesis of light-harvesting phycobilins in cyanobacteria, two members of the ferredoxin-dependent bilin reductases are involved in the reduction of the open-chain tetrapyrrole biliverdin IXα to the pink pigment phycoerythrobilin. The first reaction is catalyzed by 15,16-dihydrobiliverdin:ferredoxin oxidoreductase and produces the unstable intermediate 15,16-dihydrobiliverdin (DHBV). This intermediate is subsequently converted by phycoerythrobilin:ferredoxin oxidoreductase to the final product phycoerythrobilin. Although substrate channeling has been postulated already a decade ago, detailed experimental evidence was missing. Using a new on-column assay employing immobilized enzyme in combination with UV-Vis and fluorescence spectroscopy revealed that both enzymes transiently interact and that transfer of the intermediate is facilitated by a significantly higher binding affinity of DHBV toward phycoerythrobilin:ferredoxin oxidoreductase. Concluding from the presented data, the intermediate DHBV is transferred via proximity channeling.


Assuntos
Cianobactérias/metabolismo , Ficobilinas/biossíntese , Ficoeritrina/biossíntese , Proteínas de Bactérias/metabolismo , Biliverdina/análogos & derivados , Biliverdina/metabolismo , Cianobactérias/enzimologia , Enzimas Imobilizadas/metabolismo , Oxirredutases/metabolismo
16.
Biochemistry ; 58(46): 4610-4620, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31638374

RESUMO

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, requires iron for survival. In Mtb, MhuD is the cytosolic protein that degrades imported heme. MhuD is distinct, in both sequence and structure, from canonical heme oxygenases (HOs) but homologous with IsdG-type proteins. Canonical HO is found mainly in eukaryotes, while IsdG-type proteins are predominantly found in prokaryotes, including pathogens. While there are several published structures of MhuD and other IsdG-type proteins in complex with the heme substrate, no structures of IsdG-type proteins in complex with a product have been reported, unlike the case for HOs. We recently showed that the Mtb variant MhuD-R26S produces biliverdin IXα (αBV) rather than the wild-type mycobilin isomers. Given that mycobilin and other IsdG-type protein products like staphylobilin are difficult to isolate in quantities sufficient for structure determination, here we use the MhuD-R26S variant and its product αBV as a proxy to study the IsdG-type protein-product complex. First, we show that αBV has a nanomolar affinity for MhuD and the R26S variant. Second, we determined the MhuD-R26S-αBV complex structure to 2.5 Å, which reveals two notable features: (1) two αBV molecules bound per active site and (2) a novel α-helix (α3) that was not observed in previous MhuD-heme structures. Finally, through molecular dynamics simulations, we show that α3 is stable with the proximal αBV alone. MhuD's high affinity for the product and the observed structural and electrostatic changes that accompany substrate turnover suggest that there may be an unidentified class of proteins that are responsible for the extraction of products from MhuD and other IsdG-type proteins.


Assuntos
Proteínas de Bactérias/química , Biliverdina/metabolismo , Heme/metabolismo , Oxigenases de Função Mista/química , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/metabolismo , Biliverdina/química , Cristalografia por Raios X , Heme/química , Humanos , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Mutação Puntual , Conformação Proteica , Especificidade por Substrato , Tuberculose/microbiologia
17.
J Biol Chem ; 294(38): 13889-13901, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31366727

RESUMO

Phycobilins are light-harvesting pigments of cyanobacteria, red algae, and cryptophytes. The biosynthesis of phycoerythrobilin (PEB) is catalyzed by the subsequent action of two ferredoxin-dependent bilin reductases (FDBRs). Although 15,16-dihydrobiliverdin (DHBV):ferredoxin oxidoreductase (PebA) catalyzes the two-electron reduction of biliverdin IXα to 15,16-DHBV, PEB:ferredoxin oxidoreductase (PebB) reduces this intermediate further to PEB. Interestingly, marine viruses encode the FDBR PebS combining both activities within one enzyme. Although PebA and PebS share a canonical fold with similar substrate-binding pockets, the structural determinants for the stereo- and regiospecific modification of their tetrapyrrole substrates are incompletely understood, also because of the lack of a PebB structure. Here, we solved the X-ray crystal structures of both substrate-free and -bound PEBB from the cryptophyte Guillardia theta at 1.90 and 1.65 Å, respectively. The structures of PEBB exhibit the typical α/ß/α-sandwich fold. Interestingly, the open-chain tetrapyrrole substrate DHBV is bound in an unexpected flipped orientation within the canonical FDBR active site. Biochemical analyses of the WT enzyme and active site variants identified two central aspartate residues Asp-99 and Asp-219 as essential for catalytic activity. In addition, the conserved Arg-215 plays a critical role in substrate specificity, binding orientation, and active site integrity. Because these critical residues are conserved within certain FDBRs displaying A-ring reduction activity, we propose that they present a conserved mechanism for this reaction. The flipped substrate-binding mode indicates that two-electron reducing FDBRs utilize the same primary site within the binding pocket and that substrate orientation is the determinant for A- or D-ring regiospecificity.


Assuntos
Pigmentos Biliares/metabolismo , Oxirredutases/metabolismo , Ficoeritrina/ultraestrutura , Bacteriófagos/enzimologia , Biliverdina/química , Biliverdina/metabolismo , Catálise , Domínio Catalítico , Criptófitas/metabolismo , Cianobactérias/metabolismo , Eucariotos/metabolismo , Oxirredução , Ficobilinas/metabolismo , Ficoeritrina/metabolismo , Conformação Proteica , Especificidade por Substrato , Tetrapirróis/biossíntese
18.
ACS Chem Biol ; 14(9): 1896-1903, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31389680

RESUMO

Photo- or optoacoustics (OA) imaging is increasingly being used as a non-invasive imaging method that can simultaneously reveal structure and function in deep tissue. However, the most frequent transgenic OA labels are current fluorescent proteins that are not optimized for OA imaging. Thus, they lack OA signal strength, and their absorption maxima are positioned at short wavelengths, thus giving small penetration depths and strong background signals. Here, we apply insights from our recent determination of the structure of the fluorescent phycobiliprotein smURFP to mutate a range of residues to promote the nonradiative decay pathway that generates the OA signal. We identified hydrophobic and aromatic substitutions within the chromophore-binding pocket that substantially increase the intensity of the OA signal and red-shift the absorption. Our results demonstrate the feasibility of structure-based mutagenesis to repurpose fluorescent probes for OA imaging, and they may provide structure-function insights for de novo engineering of transgenic OA probes.


Assuntos
Proteínas de Bactérias/química , Corantes Fluorescentes/química , Imagem Óptica/métodos , Técnicas Fotoacústicas/métodos , Ficobiliproteínas/química , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biliverdina/metabolismo , Sítios de Ligação , Camundongos Nus , Mutação , Ficobiliproteínas/genética , Ficobiliproteínas/metabolismo , Ligação Proteica , Engenharia de Proteínas/métodos , Trichodesmium/química
19.
Int J Mol Sci ; 20(15)2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31344980

RESUMO

Heme oxygenase-1 (HO-1) is an intracellular enzyme that catalyzes the oxidation of heme to generate ferrous iron, carbon monoxide (CO), and biliverdin, which is subsequently converted to bilirubin. These products have anti-inflammatory, anti-oxidant, anti-apoptotic, and anti-thrombotic properties. Although HO-1 is expressed at low levels in most tissues under basal conditions, it is highly inducible in response to various pathophysiological stresses/stimuli. HO-1 induction is thus thought to be an adaptive defense system that functions to protect cells and tissues against injury in many disease settings. In atherosclerosis, HO-1 may play a protective role against the progression of atherosclerosis, mainly due to the degradation of pro-oxidant heme, the generation of anti-oxidants biliverdin and bilirubin and the production of vasodilator CO. In animal models, a lack of HO-1 was shown to accelerate atherosclerosis, whereas HO-1 induction reduced atherosclerosis. It was also reported that HO-1 induction improved the cardiac function and postinfarction survival in animal models of heart failure or myocardial infarction. Recently, we and others examined blood HO-1 levels in patients with atherosclerotic diseases, e.g., coronary artery disease (CAD) and peripheral artery disease (PAD). Taken together, these findings to date support the notion that HO-1 plays a protective role against the progression of atherosclerotic diseases. This review summarizes the roles of HO-1 in atherosclerosis and focuses on the clinical studies that examined the relationships between HO-1 levels and atherosclerotic diseases.


Assuntos
Aterosclerose/genética , Doença da Artéria Coronariana/genética , Heme Oxigenase-1/genética , Doença Arterial Periférica/genética , Animais , Aterosclerose/sangue , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Biliverdina/metabolismo , Monóxido de Carbono/metabolismo , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/patologia , Doença da Artéria Coronariana/prevenção & controle , Heme/metabolismo , Heme Oxigenase-1/uso terapêutico , Humanos , Ferro/metabolismo , Doença Arterial Periférica/sangue , Doença Arterial Periférica/patologia , Doença Arterial Periférica/prevenção & controle , Espécies Reativas de Oxigênio/metabolismo
20.
Int Immunopharmacol ; 74: 105634, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31254959

RESUMO

OBJECTIVE: Heme oxygenase-1 (HO-1) plays a critical protective role in various insults-induced acute lung injury (ALI) through its strong anti-inflammatory, anti-oxidant, and anti-apoptotic properties, but its protective role and mechanism on seawater aspiration-induced acute lung injury remains unclear. This study aimed to explore the therapeutic potential and mechanism of HO-1 to attenuate seawater aspiration-induced ALI in vivo and in vitro. METHODS: The viability and invasion of A549 cell were analyzed through cell counting kit-8 and lactate dehydrogenase release assay; the transcriptional level of inflammatory cytokines (TNF-α, IL-6, IL-8 and MCP-1) and cell proliferation-related cytokines (FoxM1, Ccnb1 and Cdc25C) in seawater-treated A549 cell were tested by qPCR; apoptotic cells were analyzed by flow cytometryd; HO-1mRNA and protein were determined by qPCR and western blotting; the fluorescent indicators (DCFH-DA, dihydroethidium, MitoSox Red and Fluo-4) were used to monitor generation of ROS and mitochondrial function. The lung wet/dry weight radio and lactate dehydrogenase activity, Sirius red staining, TUNEL assay and immunohistochemical staining with anti-pan Cytokeratin antibody were analyzed in seawater-drowning mice. The role of HO-1 on seawater-drowning pulmonary injury was explored via HO-1 activity inhibitors (Zinc protoporphyrin) in vitro and in vivo. RESULTS: Seawater exposure decreased the cellular viability, increased the production of pro-inflammatory cytokines (IL-6, IL-8 and TNF-α), induced cellular apoptosis and inhibited the expression of cell proliferation-related cytokines (FoxM1, Ccnb1 and Cdc25C). Moreover, seawater exposure led to mitochondrial dysfunction in A549 cells. Supplement of HO-1 sepcific inducer (heme) or its catalytic product (biliverdin) significantly attenuated seawater-induced A549 damage and promoted cell proliferation. However, Zinc protoporphyrin abolished the beneficial effects of HO-1 on seawater drowning-induced pulmonary tissue injury. CONCLUSION: HO-1 attenuates seawater drowning-induced lung injury by its anti-inflammatory, anti-oxidative, and anti-apoptosis function.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Afogamento/metabolismo , Heme Oxigenase-1/metabolismo , Células A549 , Animais , Biliverdina/metabolismo , Proliferação de Células , Citocinas/genética , Humanos , Inflamação/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Estresse Oxidativo , Água do Mar
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