RESUMEN
Fatty acids are used in fundamental cellular processes, such as membrane biogenesis, energy generation, post-translational modification of proteins, and so forth. These processes require the activation of fatty acids by adenosine triphosphate (ATP), followed by condensation with coenzyme-A (CoA), catalyzed by the omnipresent enzyme called Fatty acyl-CoA ligases (FACLs). However, Fatty acyl-AMP ligases (FAALs), the structural homologs of FACLs, operate in an unprecedented CoA-independent manner. FAALs transfer fatty acids to the acyl carrier protein (ACP) domain of polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) for the biosynthesis of various antibiotics, lipopeptides, virulent complex lipids, and so forth in bacteria. Recent structural and biochemical insights from our group provide a detailed understanding of the mode of CoA rejection and ACP acceptance by FAALs. In this review, we have discussed advances in the mechanistic, evolutionary, and functional understanding of FAALs and FAAL-like domains across life forms. Here, we are proposing a "Five-tier" mechanistic model to explain the specificity of FAALs. We further demonstrate how FAAL-like domains have been repurposed into a new family of proteins in eukaryotes with a novel function in lipid metabolism.
RESUMEN
Chain-length-specific subsets of diacylglycerol (DAG) lipids are proposed to regulate differential physiological responses ranging from signal transduction to modulation of the membrane properties. However, the mechanism or molecular players regulating the subsets of DAG species remain unknown. Here, we uncover the role of a conserved eukaryotic protein family, DISCO-interacting protein 2 (DIP2) as a homeostatic regulator of a chemically distinct subset of DAGs using yeast, fly, and mouse models. Genetic and chemical screens along with lipidomics analysis in yeast reveal that DIP2 prevents the toxic accumulation of specific DAGs in the logarithmic growth phase, which otherwise leads to endoplasmic reticulum stress. We also show that the fatty acyl-AMP ligase-like domains of DIP2 are essential for the redirection of the flux of DAG subspecies to storage lipid, triacylglycerols. DIP2 is associated with vacuoles through mitochondria-vacuole contact sites and such modulation of selective DAG abundance by DIP2 is found to be crucial for optimal vacuole membrane fusion and consequently osmoadaptation in yeast. Thus, the study illuminates an unprecedented DAG metabolism route and provides new insights on how cell fine-tunes DAG subspecies for cellular homeostasis and environmental adaptation.
Lipids, such as fats and hormones, constitute one of the main building blocks of cells. There are thousands of different lipids each with distinctive chemical properties that allow them to carry out specific roles. For example, a group of lipids called diacylglycerols help cells perform a myriad of tasks, like sensing external signals, making membranes, and storing energy. The production and breakdown of diacylglycerols is therefore tightly regulated. However, very little is known about the molecules involved in this metabolic process. One possible candidate is the enzyme DIP2 which is comprised of a protein module known as FAAL (short for fatty acyl-AMP ligase). FAAL belongs to a family of enzymes that synthesize lipid-like molecules in bacteria. In 2021, a group of researchers tracked the evolutionary trajectory of these bacterial proteins and found that most of them were lost in eukaryotes, such as animals and fungi. FAAL-like proteins, however, had been retained through evolution and incorporated in to DIP2. Here, Mondal, Kinatukara et al. including some of the researchers involved in the 2021 study have used a combination of genetic and biochemical experiments to investigate whether and how DIP2 contributes to lipid metabolism in eukaryotes. They found that yeast cells without the gene for DIP2 had higher levels of diacylglycerols which hampered the shape and function of certain cellular compartments. The mutant cells were also unable to convert diacylglycerols in to another group of lipids which are involved in energy storage. This effect was observed in fruit flies and mice lacking DIP2, suggesting that this role for DIP2 is conserved across most eukaryotes. Further experiments in yeast cells revealed that unlike other enzymes that metabolize diacylglycerols, DIP2 only acted on a sub-population of diacylglycerols at specific locations and times. Furthermore, yeast cells lacking DIP2 could still grow under ideal conditions, but could not cope with high or low salt concentrations in their surroundings, suggesting that the enzyme helps cells deal with environmental stresses. Since DIP2 is found in most eukaryotes, understanding how it works could be useful for multiple branches of biology. For example, some pathogenic fungi that cause diseases in crop plants and humans also rely on DIP2. Further studies are needed to better understand the role that DIP2 plays in other eukaryotic species which may shed light on other processes the enzyme is involved in.
Asunto(s)
Diglicéridos , Saccharomyces cerevisiae , Animales , Diglicéridos/metabolismo , Homeostasis , Metabolismo de los Lípidos , Ratones , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Triglicéridos/metabolismoRESUMEN
Fatty acyl-AMP ligases (FAALs) channelize fatty acids towards biosynthesis of virulent lipids in mycobacteria and other pharmaceutically or ecologically important polyketides and lipopeptides in other microbes. They do so by bypassing the ubiquitous coenzyme A-dependent activation and rely on the acyl carrier protein-tethered 4'-phosphopantetheine (holo-ACP). The molecular basis of how FAALs strictly reject chemically identical and abundant acceptors like coenzyme A (CoA) and accept holo-ACP unlike other members of the ANL superfamily remains elusive. We show that FAALs have plugged the promiscuous canonical CoA-binding pockets and utilize highly selective alternative binding sites. These alternative pockets can distinguish adenosine 3',5'-bisphosphate-containing CoA from holo-ACP and thus FAALs can distinguish between CoA and holo-ACP. These exclusive features helped identify the omnipresence of FAAL-like proteins and their emergence in plants, fungi, and animals with unconventional domain organizations. The universal distribution of FAALs suggests that they are parallelly evolved with FACLs for ensuring a CoA-independent activation and redirection of fatty acids towards lipidic metabolites.
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Acilcoenzima A/metabolismo , Adenosina Monofosfato/metabolismo , Proteínas Bacterianas/metabolismo , Ácidos Grasos/metabolismo , Ligasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Ligasas/química , Ligasas/genética , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Ideally, a bioscaffold should mimic the characteristics of an extracellular matrix of a living organ of interest. The present study deals with the formation of composite scaffolds of collagen with gum arabic. Collagen was cross-linked with oxidized gum arabic having aldehyde groups to form a porous block. By changing the oxidation level of gum arabic, incorporation of the polysaccharides into the scaffold could be varied resulting in scaffolds with variable polysaccharide to protein content. A series of scaffolds were made by altering collagen concentration and oxidation level of gum arabic. The scaffolds were tested for their physical properties, stability, biocompatibility and ability to support the cell growth. Results implied that variable polysaccharide incorporation into the scaffolds was possible depending on the oxidation level of gum arabic which could influence the swelling behavior. The scaffolds showed non-toxic behavior towards the mesenchymal stem cells and nucleus pulposa cells using viability assay in culture conditions up to 30 days; the growth of cells was seen at all combinations of gels. Nucleus pulposa cells were able to maintain their phenotype in the GACO gels. The studies show that these scaffolds are potential candidates in applications, such as tissue engineering, and can be designed to match the requirement of different cell/tissues as per their ECM.
RESUMEN
The influence of mono- and bivalent anions and micelle/submicellar clusters of different ionic and nonionic surfactants on the hydrolysis of malachite green (MG+) was studied in the presence of low alkali concentrations. Large polarizable I- ion and bivalent SO42- ion show specific interaction toward MG+ with the formation of dye-anion ion-pair resulting in a retardation of the reaction rate. Tensiometric studies for anionic surfactants sodium dodecyl sulfate (SDS) and dioctyl sodium sulfosuccinate (AOT) in the presence of dye indicate the formation of ion-pair micelles which subsequently break down to form normal micelles. A change of the absorbance value accompanied by a red shift in the electronic spectra with increase in surfactant concentration also evidenced the formation of ion-pair. The observed rate inhibition by the anionic surfactants and the interaction behavior/nature of binding between the dye and surfactant in the premicellar region have been analyzed in terms of different kinetic models. The Piszkiewicz cooperativity index (n) suggests 1:1 association between the anionic surfactant monomer and MG+ in the submicellar cluster. A catalyzing effect for the cationic surfactant, cetyltrimethylammonium bromide (CTAB), was noticed in the post-CMC region leading toward a limiting reaction rate, and the Menger-Portonoy pseudophase model was employed to explain the behavior with evaluation of the binding parameter. The nonionic surfactant poly(oxyethylene) (20) sorbitan monolaurate (Tween 20) exerts inhibition effect on the hydrolysis reaction which may be explained qualitatively by the dearth of HO- ions in the vicinity of the micelle-bound dye and quantitatively in light of the pseudophase model. Different equilibrium constants and binding constant parameters suggest appreciable binding between the dye and surfactant monomer/micelles owing to electrostatic/hydrophobic interactions.
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In this report, the dielectric nature of graphene oxide (GO) was exploited for the successful implementation of low-power pentacene thin-film transistors suitable for nonvolatile memory applications. Two different types of devices were fabricated on indium tin oxide-coated glass substrates with two different metals, viz., gold and aluminum, as the source and drain contacts. The performance of the devices was analyzed from their field-effect characteristics. Both the devices showed dominant p-type charge transport behavior. The breakdown electric field was determined to be 1.02 × 108 V/m. The current transport mechanism was explained from the output characteristics using the Fowler-Nordheim tunneling theory. Capacitance-voltage (C-V) measurements have been employed to determine the value of the oxide capacitance and to examine the memory effect. The hysteresis behavior observed from the C-V characteristics show the suitability of the device for memory applications with a low operating voltage of 3 V. The charge trapping behavior of GO was explained by the energy band diagram. Frequency-dependent C-V measurements in the range 100 kHz to 1 MHz were also performed to account for the memory window obtained in the devices. The charge retention and endurance characteristics were evaluated under a constant voltage stress to check the reliability of device operation.
RESUMEN
Thermo-alkalophilic bacterium, Geobacillus thermoleovorans secrets many enzymes including a 43â¯kDa extracellular lipase. Significant thermostability, organic solvent stability and wide substrate preferences for hydrolysis drew our attention to solve its structure by crystallography. The structure was solved by molecular replacement method and refined up to 2.14â¯Å resolution. Structure of the lipase showed an alpha-beta fold with 19 α-helices and 10 ß-sheets. The active site remains covered by a lid. One calcium and one zinc atom was found in the crystal. The structure showed a major difference (rmsd 5.6â¯Å) from its closest homolog in the amino acid region 191 to 203. Thermal unfolding of the lipase showed that the lipase is highly stable with Tm of 76⯰C. 13C NMR spectra of products upon triglyceride hydrolysate revealed that the lipase hydrolyses at both sn-1 and sn-2 positions with equal efficiency.
Asunto(s)
Proteínas Bacterianas/química , Geobacillus/enzimología , Lipasa/química , Temperatura , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Estabilidad de Enzimas , Lipasa/aislamiento & purificación , Lipasa/metabolismo , Modelos Moleculares , Conformación ProteicaRESUMEN
The oxidation of l-serine by chloro and chlorohydroxo complexes of gold(III) was spectrophotometrically investigated in acidic buffer media in the absence and presence of the anionic surfactant sodium dodecyl sulfate (SDS). The oxidation rate decreases with increase in either [H+] or [Cl-]. Gold(III) complex species react with the zwitterionic form of serine to yield acetaldehyde (principal reaction product) through oxidative decarboxylation and subsequent deamination processes. A reaction pathway involving one electron transfer from serine to Au(III) followed by homolytic cleavage of α-C-C bond with the concomitant formation of iminic cation intermediate has been proposed where Au(III) is initially reduced to Au(II). The surfactant in the submicellar region exhibits a catalytic effect on the reaction rate at [SDS] ≤ 4 mM; however, in the postmicellar region an inhibitory effect was prominent at [SDS] ≥ 4 mM. The catalytic effect below the critical micelle concentration (cmc) may be attributable to the electrostatic attraction between serine and SDS that, in turn, enhances the nucleophilicity of the carboxylate ion of the amino acid. The inhibition effect beyond cmc has been explained by considering the distribution of the reactant species between the aqueous and the micellar pseudophases that restricts the close association of the reactant species. The thermodynamic parameters Δ H0 and Δ S0 associated with the binding between serine and SDS micelle were calculated to be -14.4 ± 2 kJ mol-1 and -6.3 ± 0.5 J K-1 mol-1, respectively. Water structure rearrangement and micelle-substrate binding play instrumental roles during the transfer of the reactant species from aqueous to micellar pseudophase.
RESUMEN
Triazoles are known for their non-toxicity, higher stability and therapeutic activity. Few nucleoside (L1, L2 and L3) and non-nucleoside 1,2,3-triazoles (L4-L14) were synthesised using click chemistry and they were screened for tumor cell cytotoxicity and proliferation. Among these triazole ligands studied, nucleoside ligands exhibited higher potential than non-nucleoside ligands. The nucleoside triazole analogues, 3'-Phenyl-1,2,3- triazole-thymidine (L2) and 3'-4-Chlorophenyl-1,2,3-triazole-thymidine (L3), demonstrated higher cytotoxicity in tumor cells than in normal cells. The IC50 value for L3 was lowest (50 µM) among the ligands studied. L3 terminated cell cycle at S, G2/M phases and enhanced sub-G1 populations, manifesting induction of apoptosis in tumor cells. Confocal studies indicated that nucleoside triazole ligands (L2/L3) cause higher DNA fragmentation than other ligands. Preclinical experiments with tumor-induced mice showed greater reduction in tumor size with L3. In vitro DNA synthesis reaction with L3 exhibited higher DNA synthesis inhibition with quadruplex forming DNA (QF DNA) than non quadruplex forming DNA (NQF DNA). T(m) of quadruplex DNA increased in the presence of L3, indicating its ability to enhance stability of quadruplex DNA at elevated temperature and the results indicate that it had higher affinity towards quadruplex DNA than the other forms of DNA (like dsDNA and ssDNA). From western blot experiment, it was noticed that telomerase expression levels in the tissues of tumor-induced mice were found to be reduced on L3 treatment. Microcalorimetry results emphasise that two nucleoside triazole ligands (L2/L3) interact with quadruplex DNA with significantly higher affinity (K(d)≈10â»7 M). Interestingly the addition of an electronegative moiety to the phenyl group of L2 enhanced its anti-proliferative activity. Though IC50 values are not significantly low with L3, the studies on series of synthetic 1,2,3-triazole ligands are useful for improving and building potential pro-apoptotic ligands.
Asunto(s)
Antineoplásicos/farmacología , ADN de Neoplasias/antagonistas & inhibidores , G-Cuádruplex/efectos de los fármacos , Melanoma Experimental/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Timidina/química , Triazoles/química , Animales , Antineoplásicos/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Química Clic , Fragmentación del ADN/efectos de los fármacos , ADN de Neoplasias/biosíntesis , Humanos , Ligandos , Melanoma Experimental/química , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Neoplasias Cutáneas/química , Neoplasias Cutáneas/patología , Carga Tumoral/efectos de los fármacos , Zidovudina/químicaRESUMEN
3-Phosphoglycerate kinase (EC 2.7.2.3) is a key enzyme in the glycolytic pathway and catalyzes an important phosphorylation step leading to the production of ATP. The crystal structure of Plasmodium falciparum phosphoglycerate kinase (PfPGK) in the open conformation is presented in two different groups, namely I222 and P6(1)22. The structure in I222 space group is solved using MAD and refined at 3Å whereas that in P6(1)22A is solved using MR and refined at 2.7Å. I222 form has three monomers in asymmetric unit whereas P6(1)22 form has two monomers in the asymmetric unit. In both crystal forms a sulphate ion is located at the active site where ATP binds, but no Mg(2+) ion is observed. For the first time another sulphate ion is found at the basic patch where the 3-phosphate of 1,3-biphosphoglycerate normally binds. This was found in both chains of P6(1)22 form but only in chain A of I222 form.
Asunto(s)
Fosfoglicerato Quinasa/química , Plasmodium falciparum/enzimología , Aniones/química , Cristalografía por Rayos X , Ácidos Difosfoglicéricos/química , Magnesio/química , Unión Proteica , Conformación Proteica , Sulfatos/químicaRESUMEN
Biochanin-A, an isoflavone, existing in red clover, cabbage and alfalfa, has an inhibitory and apoptogenic effect on certain cancer cells. However, the actual mechanism by which this compound inhibits proliferation and induces apoptosis in cancer cells and the mechanism of its anti-inflammatory activities have not been well characterized. In this study, we have investigated the anti-inflammatory and anti-proliferative activity of Biochanin-A. The effects of Biochanin-A on RAW 264.7, HT-29 cell lines and mouse peritoneal macrophages have been investigated in vitro. Cell proliferation and anti-inflammatory effects were analyzed by 3-(4-5-dimethylthiozol-2-yl)2-5-diphenyl-tetrazolium bromide (MTT) assay, (3)H-thymidine incorporation assay, Western blot, cytokines estimation, Luciferase assay, Electrophoretic mobility shift assay (EMSA) and Kinase assay. Present investigation demonstrated that, Biochanin-A inhibited lipopolysacharide (LPS)-induced nitric oxide(NO) production in macrophage and showed dose dependent inhibition of inducible nitric oxide synthase (iNOS) expression. The induction of NF-κB binding activity by LPS was inhibited markedly by co-incubation with different doses of Biochanin-A. Biochanin-A inhibited the LPS-induced IkB kinase (IKK) activity and nuclear factor kappa beta (NF-κB) activation associated with the inhibition of iNOS expression. LPS-induced phosphorylation of IκBα and p38 MAPK was blocked by Biochanin-A and it inhibited IL-6, IL-1ß and TNF-α production in RAW264.7 cells indicating its anti-inflammatory activity in association with anti-proliferation. Biochanin-A is important for the prevention of phosphorylation and degradation of IκBα, thereby blocking NF-κB activation, which in turn leads to decreased expression of the iNOS, thus preventing proliferation and inflammation.
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Antiinflamatorios/farmacología , Proliferación Celular/efectos de los fármacos , Genisteína/farmacología , Inflamación/tratamiento farmacológico , Factor de Transcripción Activador 2/efectos de los fármacos , Factor de Transcripción Activador 2/metabolismo , Animales , Antiinflamatorios/administración & dosificación , Núcleo Celular/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Genisteína/administración & dosificación , Células HT29 , Humanos , Inflamación/fisiopatología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , FN-kappa B/metabolismo , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Transporte de Proteínas , Proteínas Quinasas p38 Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Soluble guanylate cyclase (sGC), a heterodimeric heme protein, catalyses the conversion of GTP in to cyclic GMP, which acts as a second messenger in cellular signaling. Nitric oxide activates this enzyme several hundred folds over its basal level. Carbon monoxide, along with some activator molecules like YC-1 and BAY, also synergistically activate sGC. Mechanism of this synergistic activation is a matter of debate. Here we review the existing literature to identify the possible binding site for YC-1 and BAY on bovine lung sGC and its mechanism of activation. These two exogenous compounds bind sGC on alpha subunit inside a pocket and thus exert allosteric effect via subunit interface, which is relayed to the catalytic site. We used docking studies to further validate this hypothesis. We propose that the binding of YC-1/BAY inside the sensory domain of the alpha subunit modulates the interactions on the subunit interface resulting in rearrangements in the catalytic site into active conformation and this partly induces the cleavage of Fe-His bond.
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Furanos/metabolismo , Guanilato Ciclasa/metabolismo , Indazoles/metabolismo , Pirazoles/metabolismo , Piridinas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Regulación Alostérica , Animales , Sitios de Unión , Bovinos , Sinergismo Farmacológico , Activación Enzimática , Furanos/farmacología , Guanilato Ciclasa/química , Indazoles/farmacología , Pulmón/enzimología , Modelos Químicos , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Pirazoles/farmacología , Piridinas/farmacología , Receptores Citoplasmáticos y Nucleares/química , Guanilil Ciclasa SolubleRESUMEN
Proteins belonging to the lipocalin superfamily are usually secretory proteins of molecular mass approximately 20 kDa with a hydrophobic pocket for the binding and transport of diverse small ligands. Various lipocalins have been associated with many biological processes, e.g. immunomodulation, odorant transport, pheromonal activity, retinoid transport, cancer-cell interactions etc. However, the exact functions of many lipocalins and the ligands bound by them are unclear. Previously, the cDNA of a 20 kDa lipocalin (FLP) which is female-specifically expressed in the lacrimal glands of Syrian (golden) hamsters and secreted in the tears of females has been identified and cloned. His-tagged recombinant FLP (rFLP) has now been cloned, overexpressed in Escherichia coli as a soluble protein and purified to homogeneity using Ni-affinity followed by size-exclusion chromatography. Purified rFLP was crystallized using the sitting-drop vapour-diffusion method. The crystals tested belonged to space group P2(1)2(1)2(1) and diffracted to beyond 1.86 A resolution. Solvent-content analysis indicated the presence of one monomer in the asymmetric unit.
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Aparato Lagrimal/química , Lipocalinas/química , Mesocricetus , Animales , Clonación Molecular , Cricetinae , Cristalización , Cristalografía por Rayos X , Femenino , Expresión Génica , Lipocalinas/genética , Lipocalinas/aislamiento & purificaciónRESUMEN
To ensure a high fidelity during translation, threonyl-tRNA synthetases (ThrRSs) harbor an editing domain that removes noncognate L-serine attached to tRNAThr. Most archaeal ThrRSs possess a unique editing domain structurally similar to D-aminoacyl-tRNA deacylases (DTDs) found in eubacteria and eukaryotes that specifically removes D-amino acids attached to tRNA. Here, we provide mechanistic insights into the removal of noncognate L-serine from tRNAThr by a DTD-like editing module from Pyrococcus abyssi ThrRS (Pab-NTD). High-resolution crystal structures of Pab-NTD with pre- and post-transfer substrate analogs and with L-serine show mutually nonoverlapping binding sites for the seryl moiety. Although the pre-transfer editing is excluded, the analysis reveals the importance of main chain atoms in proper positioning of the post-transfer substrate for its hydrolysis. A single residue has been shown to play a pivotal role in the inversion of enantioselectivity both in Pab-NTD and DTD. The study identifies an enantioselectivity checkpoint that filters opposite chiral molecules and thus provides a fascinating example of how nature has subtly engineered this domain for the selection of chiral molecules during translation.
Asunto(s)
Proteínas Arqueales/química , Modelos Moleculares , Pyrococcus abyssi/enzimología , Edición de ARN , Aminoacil-ARN de Transferencia/química , Treonina-ARNt Ligasa/química , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Sitios de Unión , Lisina/química , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Pyrococcus abyssi/genética , Aminoacil-ARN de Transferencia/genética , Estereoisomerismo , Treonina-ARNt Ligasa/genética , Aminoacilación de ARN de TransferenciaRESUMEN
Gas sensory heme proteins respond to their environment by binding a specific gas molecule to heme and transmitting this primary binding signal to the protein. How the binding signal is transmitted from the heme to the protein remains to be clarified. Using UV resonance Raman (UVRR) spectroscopy, we investigated this pathway in sperm whale myoglobin as a model gas sensory heme protein. Based on the UVRR data and the effects of deleting one of three important pathways (His-93, 6-propionate, or 7-propionate), we determined the changes in the conformation of globin that occur upon binding of CO, nitric oxide (NO), or O(2) to heme and how they are transmitted from heme to globin. The UVRR results show that heme discriminates different ligands, resulting in different conformations in the globin protein. Specifically, NO induces changes in the spectrum of Trp residues in the A-helix that are significantly different from those induced by O(2) or CO binding. On the other hand, binding of O(2) to heme produces changes in the Tyr residues of the H-helix that are different from those induced by CO or NO binding. Furthermore, we found that cleavage of the Fe-His-93 covalent bond eliminates communication to the terminal region of the H-helix and that the 7-propionate hydrogen-bonding network is essential for transmitting the CO or NO binding signal to the N and C termini. Finally, the 6-propionate is important only for NO binding. Thus, the hydrogen-bonding network in the protein appears to be critical for intramolecular signal transduction in gas sensory heme proteins.
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Hemo/química , Mioglobina/química , Animales , Caballos , Enlace de Hidrógeno , Modelos Moleculares , Conformación Molecular , Espectrometría Raman , Rayos UltravioletaRESUMEN
Aldoxime dehydratase (Oxd) is a novel hemeprotein that catalyzes the dehydration reaction of aldoxime to produce nitrile. In this study, we studied the spectroscopic and substrate binding properties of two Oxds, OxdB from Bacillus sp. strain OxB-1 and OxdRE from Rhodococcus sp. N-771, that show different quaternary structures and relatively low amino acid sequence identity. Electronic absorption and resonance Raman spectroscopy revealed that ferric OxdRE contained a six-coordinate low-spin heme, while ferric OxdB contained a six-coordinate high-spin heme. Both ferrous OxdRE and OxdB included a five-coordinate high-spin heme to which the substrate was bound via its nitrogen atom for the reaction to occur. Although the ferric Oxds were inactive for catalysis, the substrate was bound to the ferric heme via its oxygen atom in both OxdB and OxdRE. Electronic paramagnetic resonance (EPR) and rapid scanning spectroscopy revealed that the flexibility of the heme pocket was different between OxdB and OxdRE, which might affect their substrate specificity.
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Hemo/química , Hidroliasas/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Hidroliasas/química , Espectrofotometría Ultravioleta , Espectrometría Raman , Especificidad por SustratoRESUMEN
Reduced pteridines are required for a number of important cellular functions. Trypanosomatid parasites, unlike their mammalian hosts, are pteridine auxotrophs and salvage the precursor pteridines from the host and reduce them to the respective biologically active tetrahydro forms using parasite-encoded enzymes. These enzymes may offer selective drug targets. In Leishmania, pteridine reductase 1 (PTR1), the primary enzyme for reducing pterins, is also responsible for resistance to antifolate drugs. Typically, PTR1 is more active with fully oxidized biopterin and folate than with their reduced counterparts. We have identified an enzyme, TcPTR2 of Trypanosoma cruzi, which though very similar to PTR1 in its primary sequence, can reduce only dihydrobiopterin and dihydrofolate and not oxidized pteridines. The structures of an inhibitor (methotrexate) and a substrate (dihydrofolate) complex of this enzyme demonstrate that the orientation of the substrate and the inhibitor in the active site of TcPTR2 are different from each other. However, the orientation of each ligand is similar to that of the corresponding ligand in Leishmania major PTR1 complexes.
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Isoenzimas/química , Oxidorreductasas/química , Estructura Cuaternaria de Proteína , Proteínas Protozoarias/química , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Ácido Fólico/análogos & derivados , Ácido Fólico/química , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Sustancias Macromoleculares , Metotrexato/química , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , NADP/química , Oxidorreductasas/genética , Oxidorreductasas/aislamiento & purificación , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Alineación de SecuenciaRESUMEN
Resonance Raman (RR) spectra of soluble guanylate cyclase (sGC) reported by five independent research groups have been classified as two types: sGC(1) and sGC(2). Here we demonstrate that the RR spectra of sGC isolated from bovine lung contain only sGC(2) while both species are observed in the spectra of the CO-bound form (CO-sGC). The relative populations of the two forms were altered from an initial composition in which the CO-sGC(2) form predominated, with the Fe-CO (nu(Fe)(-)(CO)) and C-O stretching modes (nu(CO)) at 472 and 1985 cm(-)(1), respectively, to a composition dominated by the CO-sGC(1) form with nu(Fe)(-)(CO) and nu(CO) at 488 and 1969 cm(-)(1), respectively, following the addition of a xenobiotic, YC-1. Further addition of a substrate, GTP, completed the change. GDP and cGMP had a significantly weaker effect, while a substrate analogue, GTP-gamma-S, had an effect similar to that of GTP. In contrast, ATP had a reverse effect, and suppressed the effects of YC-1 and GTP. In the presence of both YC-1 and GTP, vinyl vibrations of heme were significantly influenced. New CO isotope-sensitive bands were observed at 521, 488, 363, and 227 cm(-)(1). The 521 cm(-)(1) band was assigned to the five-coordinate (5c) species from the model compound studies using ferrous iron protoporphyrin IX in CTAB micelles. Distinct from the 472 cm(-)(1) species, both the 488 and 521 cm(-)(1) species were apparently un-photodissociable when an ordinary Raman spinning cell was used, indicating rapid recombination of photodissociated CO. On the basis of these findings, binding of YC-1 to the heme pocket is proposed.
Asunto(s)
Monóxido de Carbono/metabolismo , Guanilato Ciclasa/metabolismo , Hemo/metabolismo , Regulación Alostérica , Animales , Bovinos , Nucleótidos de Guanina/metabolismo , Unión Proteica , Espectrometría RamanRESUMEN
Soluble guanylate cyclase (sGC, EC 4.6.1.2) acts as a sensor for nitric oxide (NO), but is also activated by carbon monoxide in the presence of an allosteric modulator. Resonance Raman studies on the structure-function relations of sGC are reviewed with a focus on the CO-adduct in the presence and absence of allosteric modulator, YC-1, and substrate analogues. It is demonstrated that the sGC isolated from bovine lung contains one species with a five-coordinate (5c) ferrous high-spin heme with the Fe-His stretching mode at 204 cm(-1), but its CO adduct yields two species with different conformations about the heme pocket with the Fe-CO stretching (nuFe-CO) mode at 473 and 489 cm(-1), both of which are His- and CO-coordinated 6c ferrous adducts. Addition of YC-1 to it changes their population and further addition of GTP yields one kind of 6c (nuFe-CO=489 cm(-1)) in addition to 5c CO-adduct (nuFe-CO=521 cm(-1)). Under this condition the enzymatic activity becomes nearly the same level as that of NO adduct. Addition of gamma-S-GTP yields the same effect as GTP does but cGMP and GDP gives much less effects. Unexpectedly, ATP cancels the effects of GTP. The structural meaning of these spectroscopic observations is discussed in detail.
Asunto(s)
Monóxido de Carbono/metabolismo , Guanilato Ciclasa/química , Guanilato Ciclasa/metabolismo , Óxido Nítrico/metabolismo , Regulación Alostérica , Animales , Dominio Catalítico , Activación Enzimática , Modelos Moleculares , Estructura Molecular , Estructura Terciaria de Proteína , Espectrometría RamanRESUMEN
While most of CO-bound hemes are easily photodissociated with a quantum yield of nearly unity, we occasionally encounter a CO-heme which appears hardly photodissociable under the ordinary measurement conditions of resonance Raman spectra using CW laser excitation and a spinning cell. This study aims to understand such hemes theoretically, that is, the excited-state properties of the five-coordinate heme-CO adduct (5cH) as well as the 6c heme-CO adduct (6cH) with a weak axial ligand. Using a hybrid density functional theory, we scrutinized the properties of the ground and excited spin states of the computational models of a 5cH and a water-ligated 6cH (6cH-H(2)O) and compared these properties with those of a photodissociable imidazole-ligated 6cH (6cH-Im). Jahn-Teller softening for the Fe-C-O bending potential in the a(1)-e excited state was suggested. The excited-state properties of 6cH-Im and 5cH were further studied with time-dependent DFT theory. The reaction products of 6cH-Im and 5cH were assumed to be quintet and triplet states, respectively. According to the time-dependent DFT calculations, the Q excited state of 6cH-Im, which is initially a pure pi-pi state, crosses the Fe-CO dissociative state (2A') without large elongation of the Fe-CO bond. In contrast, the Q state of the 5cH does not cross the Fe-CO dissociative state but results in the formation of the excited spin state with a bent Fe-C-O. Consequently, photoisomerization from linear to bent Fe-C-O in the 5cH is a likely mechanism for apparent nonphotodissociation.