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1.
Mol Cell ; 65(3): 377-379, 2017 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-28157502

RESUMEN

Adaptation of CRISPR-Cas9 for genome-editing applications has revolutionized biomedical research. New single-component effector CRISPR systems are emerging from the bioinformatics pipeline. How can we best harness their power? Three new studies will no doubt facilitate this transition by generating the C2c1 and C2c2 structure snapshots in different functional states.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Proteínas Asociadas a CRISPR/química , Ingeniería Genética/métodos , Modelos Moleculares
2.
Plant Physiol ; 183(3): 957-973, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32332088

RESUMEN

Cinnamate 4-hydroxylase (C4H; CYP73A) is a cytochrome P450 monooxygenase associated externally with the endoplasmic reticulum of plant cells. The enzyme uses NADPH-cytochrome P450 reductase as a donor of electrons and hydroxylates cinnamic acid to form 4-coumaric acid in phenylpropanoid metabolism. In order to better understand the structure and function of this unique class of plant P450 enzymes, we have characterized the enzyme C4H1 from lignifying tissues of sorghum (Sorghum bicolor), encoded by Sobic.002G126600 Here we report the 1.7 Å resolution crystal structure of CYP73A33. The obtained structural information, along with the results of the steady-state kinetic analysis and the absorption spectroscopy titration, displays a high degree of similarity of the structural and functional features of C4H to those of other P450 proteins. Our data also suggest the presence of a putative allosteric substrate-binding site in a hydrophobic pocket on the enzyme surface. In addition, comparing the newly resolved structure with those of well-investigated cytochromes P450 from mammals and bacteria enabled us to identify those residues of critical functional importance and revealed a unique sequence signature that is potentially responsible for substrate specificity and catalytic selectivity of C4H.


Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Regulación de la Expresión Génica de las Plantas , Sorghum/genética , Sorghum/metabolismo , Transcinamato 4-Monooxigenasa/genética , Transcinamato 4-Monooxigenasa/metabolismo , Genes de Plantas , Estructura Molecular
3.
Mol Microbiol ; 112(6): 1863-1874, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31580513

RESUMEN

Ethylenediaminetetraacetate (EDTA) is the most abundant organic pollutant in surface water because of its extensive usage and the recalcitrance of stable metal-EDTA complexes. A few bacteria including Chelativorans sp. BNC1 can degrade EDTA with a monooxygenase to ethylenediaminediacetate (EDDA) and then use iminodiacetate oxidase (IdaA) to further degrade EDDA into ethylenediamine in a two-step oxidation. To alleviate EDTA pollution into the environment, deciphering the mechanisms of the metabolizing enzymes is an imperative prerequisite for informed EDTA bioremediation. Although IdaA cannot oxidize glycine, the crystal structure of IdaA shows its tertiary and quaternary structures similar to those of glycine oxidases. All confirmed substrates, EDDA, ethylenediaminemonoacetate, iminodiacetate and sarcosine are secondary amines with at least one N-acetyl group. Each substrate was bound at the re-side face of the isoalloxazine ring in a solvent-connected cavity. The carboxyl group of the substrate was bound by Arg265 and Arg307 . The catalytic residue, Tyr250 , is under the hydrogen bond network to facilitate its deprotonation acting as a general base, removing an acetate group of secondary amines as glyoxylate. Thus, IdaA is a secondary amine oxidase, and our findings improve understanding of molecular mechanism involved in the bioremediation of EDTA and the metabolism of secondary amines.


Asunto(s)
Ácido Edético/metabolismo , Monoaminooxidasa , Phyllobacteriaceae/enzimología , Aminas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biodegradación Ambiental , Quelantes del Calcio/metabolismo , Cristalografía por Rayos X , Contaminantes Ambientales/metabolismo , Monoaminooxidasa/química , Monoaminooxidasa/metabolismo
4.
Int J Mol Sci ; 21(11)2020 May 30.
Artículo en Inglés | MEDLINE | ID: mdl-32486296

RESUMEN

The widespread use of synthetic aminopolycarboxylates, such as ethylenediaminetetraacetate (EDTA), as chelating agents has led to their contamination in the environment as stable metal-chelate complexes. Microorganisms can transport free EDTA, but not metal-EDTA complexes, into cells for metabolism. An ABC-type transporter for free EDTA uptake in Chelativorans sp. BNC1 was investigated to understand the mechanism of the ligand selectivity. We solved the X-ray crystal structure of the periplasmic EDTA-binding protein (EppA) and analyzed its structure-function relations through isothermal titration calorimetry, site-directed mutagenesis, molecular docking, and quantum chemical analysis. EppA had high affinities for EDTA and other aminopolycarboxylates, which agrees with structural analysis, showing that its binding pocket could accommodate free aminopolycarboxylates. Further, key amino acid residues involved in the binding were identified. Our results suggest that EppA is a general binding protein for the uptake of free aminopolycarboxylates. This finding suggests that bacterial cells import free aminopolycarboxylates, explaining why stable metal-chelate complexes are resistant to degradation, as they are not transported into the cells for degradation.


Asunto(s)
Proteínas Bacterianas/metabolismo , Ácidos Carboxílicos/metabolismo , Ácido Edético/química , Proteínas de Unión Periplasmáticas/metabolismo , Phyllobacteriaceae/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Calorimetría , Quelantes/química , Cristalografía por Rayos X , Ligandos , Luz , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación Proteica , Dominios Proteicos , Dispersión de Radiación , Electricidad Estática , Termodinámica
5.
Biochemistry ; 58(38): 3960-3970, 2019 09 24.
Artículo en Inglés | MEDLINE | ID: mdl-31469273

RESUMEN

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR), in most organisms, catalyzes the four-electron reduction of the thioester (S)-HMG-CoA to the primary alcohol (R)-mevalonate, utilizing NADPH as the hydride donor. In some organisms, including the opportunistic lung pathogen Burkholderia cenocepacia, it catalyzes the reverse reaction, utilizing NAD+ as a hydride acceptor in the oxidation of mevalonate. B. cenocepacia HMGR has been previously shown to exist as an ensemble of multiple non-additive oligomeric states, each with different levels of enzymatic activity, suggesting that the enzyme exhibits characteristics of the morpheein model of allostery. We have characterized a number of factors, including pH, substrate concentration, and enzyme concentration, that modulate the structural transitions that influence the interconversion among the multiple oligomers. We have also determined the crystal structure of B. cenocepacia HMGR in the hexameric state bound to coenzyme A and ADP. This hexameric assembly provides important clues about how the transition among oligomers might occur, and why B. cenocepacia HMGR, unique among characterized HMGRs, exhibits morpheein-like behavior.


Asunto(s)
Proteínas Bacterianas/metabolismo , Burkholderia cenocepacia/enzimología , Hidroximetilglutaril-CoA Reductasas/metabolismo , Estructura Cuaternaria de Proteína , Adenosina Trifosfato/química , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Coenzima A/química , Cristalografía por Rayos X , Pruebas de Enzimas , Hidroximetilglutaril-CoA Reductasas/química , Hidroximetilglutaril-CoA Reductasas/aislamiento & purificación , Simulación de Dinámica Molecular , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo
6.
Plant Physiol ; 173(1): 417-433, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27879392

RESUMEN

Class III peroxidases (CIIIPRX) catalyze the oxidation of monolignols, generate radicals, and ultimately lead to the formation of lignin. In general, CIIIPRX genes encode a large number of isozymes with ranges of in vitro substrate specificities. In order to elucidate the mode of substrate specificity of these enzymes, we characterized one of the CIIIPRXs (PviPRX9) from switchgrass (Panicum virgatum), a strategic plant for second-generation biofuels. The crystal structure, kinetic experiments, molecular docking, as well as expression patterns of PviPRX9 across multiple tissues and treatments, along with its levels of coexpression with the majority of genes in the monolignol biosynthesis pathway, revealed the function of PviPRX9 in lignification. Significantly, our study suggested that PviPRX9 has the ability to oxidize a broad range of phenylpropanoids with rather similar efficiencies, which reflects its role in the fortification of cell walls during normal growth and root development and in response to insect feeding. Based on the observed interactions of phenylpropanoids in the active site and analysis of kinetics, a catalytic mechanism involving two water molecules and residues histidine-42, arginine-38, and serine-71 was proposed. In addition, proline-138 and gluntamine-140 at the 137P-X-P-X140 motif, leucine-66, proline-67, and asparagine-176 may account for the broad substrate specificity of PviPRX9. Taken together, these observations shed new light on the function and catalysis of PviPRX9 and potentially benefit efforts to improve biomass conservation properties in bioenergy and forage crops.


Asunto(s)
Panicum/enzimología , Peroxidasas/química , Peroxidasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Biocatálisis , Calcio/metabolismo , Cristalografía por Rayos X , Pruebas de Enzimas , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Hemo/metabolismo , Peróxido de Hidrógeno/metabolismo , Cinética , Funciones de Verosimilitud , Metaboloma , Simulación del Acoplamiento Molecular , Panicum/genética , Peroxidasas/genética , Estructura Secundaria de Proteína , Electricidad Estática , Especificidad por Sustrato
7.
Mol Microbiol ; 100(6): 989-1003, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26928990

RESUMEN

Ethylenediaminetetraacetate (EDTA) is currently the most abundant organic pollutant due to its recalcitrance and extensive use. Only a few bacteria can degrade it, using EDTA monooxygenase (EmoA) to initiate the degradation. EmoA is an FMNH2 -dependent monooxygenase that requires an NADH:FMN oxidoreductase (EmoB) to provide FMNH2 as a cosubstrate. Although EmoA has been identified from Chelativorans (ex. Mesorhizobium) sp. BNC1, its catalytic mechanism is unknown. Crystal structures of EmoA revealed a domain-like insertion into a TIM-barrel, which might serve as a flexible lid for the active site. Docking of MgEDTA(2-) into EmoA identified an intricate hydrogen bond network connected to Tyr(71) , which should potentially lower its pKa. Tyr(71) , along with nearby Glu(70) and a peroxy flavin, facilitates a keto-enol transition of the leaving acetyl group of EDTA. Further, for the first time, the physical interaction between EmoA and EmoB was observed by ITC, molecular docking and enzyme kinetic assay, which enhanced both EmoA and EmoB activities probably through coupled channelling of FMNH2 .


Asunto(s)
FMN Reductasa/química , FMN Reductasa/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Secuencia de Aminoácidos , Catálisis , Dominio Catalítico , Ácido Edético/metabolismo , Mononucleótido de Flavina/metabolismo , Flavinas/metabolismo , Hidroquinonas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , NADH NADPH Oxidorreductasas/metabolismo , Oxidorreductasas/metabolismo , Phyllobacteriaceae/enzimología , Phyllobacteriaceae/metabolismo , Relación Estructura-Actividad
8.
J Biol Chem ; 290(48): 28665-74, 2015 Nov 27.
Artículo en Inglés | MEDLINE | ID: mdl-26416891

RESUMEN

Calsequestrin 1 is the principal Ca(2+) storage protein of the sarcoplasmic reticulum of skeletal muscle. Its inheritable D244G mutation causes a myopathy with vacuolar aggregates, whereas its M87T "variant" is weakly associated with malignant hyperthermia. We characterized the consequences of these mutations with studies of the human proteins in vitro. Equilibrium dialysis and turbidity measurements showed that D244G and, to a lesser extent, M87T partially lose Ca(2+) binding exhibited by wild type calsequestrin 1 at high Ca(2+) concentrations. D244G aggregates abruptly and abnormally, a property that fully explains the protein inclusions that characterize its phenotype. D244G crystallized in low Ca(2+) concentrations lacks two Ca(2+) ions normally present in wild type that weakens the hydrophobic core of Domain II. D244G crystallized in high Ca(2+) concentrations regains its missing ions and Domain II order but shows a novel dimeric interaction. The M87T mutation causes a major shift of the α-helix bearing the mutated residue, significantly weakening the back-to-back interface essential for tetramerization. D244G exhibited the more severe structural and biophysical property changes, which matches the different pathophysiological impacts of these mutations.


Asunto(s)
Proteínas de Unión al Calcio/química , Calcio/química , Hipertermia Maligna , Proteínas Mitocondriales/química , Enfermedades Musculares , Mutación Missense , Sustitución de Aminoácidos , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Calsecuestrina , Cristalografía por Rayos X , Humanos , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína
9.
J Biol Chem ; 290(31): 18914-23, 2015 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-26082492

RESUMEN

Persulfide dioxygenases (PDOs), also known as sulfur dioxygenases (SDOs), oxidize glutathione persulfide (GSSH) to sulfite and GSH. PDOs belong to the metallo-ß-lactamase superfamily and play critical roles in animals, plants, and microorganisms, including sulfide detoxification. The structures of two PDOs from human and Arabidopsis thaliana have been reported; however, little is known about the substrate binding and catalytic mechanism. The crystal structures of two bacterial PDOs from Pseudomonas putida and Myxococcus xanthus were determined at 1.5- and 2.5-Å resolution, respectively. The structures of both PDOs were homodimers, and their metal centers and ß-lactamase folds were superimposable with those of related enzymes, especially the glyoxalases II. The PDOs share similar Fe(II) coordination and a secondary coordination sphere-based hydrogen bond network that is absent in glyoxalases II, in which the corresponding residues are involved instead in coordinating a second metal ion. The crystal structure of the complex between the Pseudomonas PDO and GSH also reveals the similarity of substrate binding between it and glyoxalases II. Further analysis implicates an identical mode of substrate binding by known PDOs. Thus, the data not only reveal the differences in metal binding and coordination between the dioxygenases and the hydrolytic enzymes in the metallo-ß-lactamase superfamily, but also provide detailed information on substrate binding by PDOs.


Asunto(s)
Proteínas Bacterianas/química , Dioxigenasas/química , Myxococcus xanthus/enzimología , Pseudomonas putida/enzimología , beta-Lactamasas/química , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Glutatión , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Soluciones , Especificidad por Sustrato
10.
Int J Mol Sci ; 17(9)2016 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-27649144

RESUMEN

Calsequestrin is glycosylated and phosphorylated during its transit to its final destination in the junctional sarcoplasmic reticulum. To determine the significance and universal profile of these post-translational modifications to mammalian calsequestrin, we characterized, via mass spectrometry, the glycosylation and phosphorylation of skeletal muscle calsequestrin from cattle (B. taurus), lab mice (M. musculus) and lab rats (R. norvegicus) and cardiac muscle calsequestrin from cattle, lab rats and humans. On average, glycosylation of skeletal calsequestrin consisted of two N-acetylglucosamines and one mannose (GlcNAc2Man1), while cardiac calsequestrin had five additional mannoses (GlcNAc2Man6). Skeletal calsequestrin was not phosphorylated, while the C-terminal tails of cardiac calsequestrin contained between zero to two phosphoryls, indicating that phosphorylation of cardiac calsequestrin may be heterogeneous in vivo. Static light scattering experiments showed that the Ca(2+)-dependent polymerization capabilities of native bovine skeletal calsequestrin are enhanced, relative to the non-glycosylated, recombinant isoform, which our crystallographic studies suggest may be due to glycosylation providing a dynamic "guiderail"-like scaffold for calsequestrin polymerization. Glycosylation likely increases a polymerization/depolymerization response to changing Ca(2+) concentrations, and proper glycosylation, in turn, guarantees both effective Ca(2+) storage/buffering of the sarcoplasmic reticulum and localization of calsequestrin (Casq) at its target site.


Asunto(s)
Calsecuestrina/metabolismo , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Procesamiento Proteico-Postraduccional , Acetilglucosamina/metabolismo , Animales , Calcio/metabolismo , Bovinos , Glicosilación , Manosa/metabolismo , Ratones , Fosforilación , Ratas
11.
Plant Physiol ; 165(4): 1440-1456, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24948836

RESUMEN

Using S-adenosyl-methionine as the methyl donor, caffeic acid O-methyltransferase from sorghum (Sorghum bicolor; SbCOMT) methylates the 5-hydroxyl group of its preferred substrate, 5-hydroxyconiferaldehyde. In order to determine the mechanism of SbCOMT and understand the observed reduction in the lignin syringyl-to-guaiacyl ratio of three brown midrib12 mutants that carry COMT gene missense mutations, we determined the apo-form and S-adenosyl-methionine binary complex SbCOMT crystal structures and established the ternary complex structure with 5-hydroxyconiferaldehyde by molecular modeling. These structures revealed many features shared with monocot ryegrass (Lolium perenne) and dicot alfalfa (Medicago sativa) COMTs. SbCOMT steady-state kinetic and calorimetric data suggest a random bi-bi mechanism. Based on our structural, kinetic, and thermodynamic results, we propose that the observed reactivity hierarchy among 4,5-dihydroxy-3-methoxycinnamyl (and 3,4-dihydroxycinnamyl) aldehyde, alcohol, and acid substrates arises from the ability of the aldehyde to stabilize the anionic intermediate that results from deprotonation of the 5-hydroxyl group by histidine-267. Additionally, despite the presence of other phenylpropanoid substrates in vivo, sinapaldehyde is the preferential product, as demonstrated by its low Km for 5-hydroxyconiferaldehyde. Unlike its acid and alcohol substrates, the aldehydes exhibit product inhibition, and we propose that this is due to nonproductive binding of the S-cis-form of the aldehydes inhibiting productive binding of the S-trans-form. The S-cis-aldehydes most likely act only as inhibitors, because the high rotational energy barrier around the 2-propenyl bond prevents S-trans-conversion, unlike alcohol substrates, whose low 2-propenyl bond rotational energy barrier enables rapid S-cis/S-trans-interconversion.

12.
J Surg Res ; 193(2): 825-30, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25266604

RESUMEN

BACKGROUND: Microporous Polysaccharide Hemospheres (MPH) are a new plant-derived polysaccharide powder hemostat. Previous studies investigated MPH as a replacement to nonflowable hemostatic agents of different application techniques (e.g., oxidized cellulose, collagen); therefore, the purpose of this study was to determine if MPH is a surrogate for flowable hemostatic agents of similar handling and application techniques, specifically a flowable thrombin-gelatin hemostatic matrix. METHODS: Hemostatic efficacy was compared using a heparinized porcine abrasion model mimicking a capsular tear of a parenchymal organ. MPH (ARISTA, 1 g) and hemostatic matrix (Floseal, 1 mL) were applied, according to a randomized scheme, to paired hepatic abrasions (40 lesions per group). Hemostatic success, control of bleeding, and blood loss were assessed 2, 5, and 10 min after treatment. Hemostatic success and control of bleeding were analyzed using odds ratios and blood loss using mean differences. RESULTS: Hemostatic matrix provided superior hemostatic success relative to MPH at 5 (odds ratio: 0.035, 95% confidence interval: 0.004-0.278) and 10 min (0.032, 0.007-0.150), provided superior control of bleeding at 5 (0.006, <0.001-0.037) and 10 min (0.009, 0.001-0.051), and had significantly less blood loss at 5 (mean difference: 0.3118 mL/min, 95% confidence interval: 0.0939-0.5296) and 10 min (0.5025, 0.2489-0.7561). CONCLUSIONS: These findings corroborate other MPH investigations regarding its low-level efficacy and suggest that MPH is not an appropriate surrogate for hemostatic matrix despite similar application techniques. The lack of a procoagulant within MPH may likely be the reason for its lower efficacy and need for multiple applications.


Asunto(s)
Traumatismos Abdominales/terapia , Esponja de Gelatina Absorbible/uso terapéutico , Hemostasis Quirúrgica , Hígado/lesiones , Almidón/uso terapéutico , Animales , Bovinos , Femenino , Humanos , Distribución Aleatoria , Porcinos
13.
J Biol Chem ; 288(40): 28447-56, 2013 Oct 04.
Artículo en Inglés | MEDLINE | ID: mdl-23955343

RESUMEN

TftG, 5-chloro-2-hydroxyhydroquinone (5-CHQ) dehydrochlorinase, is involved in the biodegradation of 2,4,5-trichlorophenoxyacetate by Burkholderia phenoliruptrix AC1100. It belongs to the YCII superfamily, a group of proteins with largely unknown function. In this work, we utilized structural and functional studies, including the apo-form and 2,5-dihydroxybenzoquinone binary complex crystal structures, computational analysis, and site-directed mutagenesis, to determine the dehydrochlorination mechanism. The His-Asp dyad, which initiates catalysis, is strongly conserved in YCII-like proteins. In addition, other catalytically important residues such as Pro-76, which orients the His-Asp catalytic dyad; Arg-17 and Ser-56, which form an oxyanion hole; and Asp-9, which stabilizes the oxyanion hole, are among the most highly conserved residues across the YCII superfamily members. The comprehensive characterization of TftG helps not only for identifying effective mechanisms for chloroaromatic dechlorination but also for understanding the functions of YCII superfamily members, which we propose to be lyases.


Asunto(s)
Biocatálisis , Burkholderia/enzimología , Hidroquinonas/metabolismo , Liasas/metabolismo , Familia de Multigenes , Secuencia de Aminoácidos , Dominio Catalítico , Dicroismo Circular , Cristalografía por Rayos X , Hidroquinonas/química , Ligandos , Liasas/química , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Estructura Cuaternaria de Proteína , Teoría Cuántica , Alineación de Secuencia , Soluciones , Especificidad por Sustrato
14.
Mol Microbiol ; 88(3): 523-36, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23489289

RESUMEN

PcpA (2,6-dichloro-p-hydroquinone 1,2-dioxygenase) from Sphingobium chlorophenolicum, a non-haem Fe(II) dioxygenase capable of cleaving the aromatic ring of p-hydroquinone and its substituted variants, is a member of the recently discovered p-hydroquinone 1,2-dioxygenases. Here we report the 2.6 Å structure of PcpA, which consists of four ßαßßß motifs, a hallmark of the vicinal oxygen chelate superfamily. The secondary co-ordination sphere of the Fe(II) centre forms an extensive hydrogen-bonding network with three solvent exposed residues, linking the catalytic Fe(II) to solvent. A tight hydrophobic pocket provides p-hydroquinones access to the Fe(II) centre. The p-hydroxyl group is essential for the substrate-binding, thus phenols and catechols, lacking a p-hydroxyl group, do not bind to PcpA. Site-directed mutagenesis and kinetic analysis confirm the critical catalytic role played by the highly conserved His10, Thr13, His226 and Arg259. Based on these results, we propose a general reaction mechanism for p-hydroquinone 1,2-dioxygenases.


Asunto(s)
Dioxigenasas/química , Sphingomonadaceae/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Calorimetría , Catálisis , Catecoles/farmacología , Compuestos Ferrosos , Hidroquinonas/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Alineación de Secuencia , Sphingomonadaceae/genética
15.
Int J Mol Sci ; 15(11): 20736-52, 2014 Nov 12.
Artículo en Inglés | MEDLINE | ID: mdl-25397598

RESUMEN

PcpR is a LysR-type transcription factor from Sphingobium chlorophenolicum L-1 that is responsible for the activation of several genes involved in polychlorophenol degradation. PcpR responds to several polychlorophenols in vivo. Here, we report the crystal structures of the inducer-binding domain of PcpR in the apo-form and binary complexes with pentachlorophenol (PCP) and 2,4,6-trichlorophenol (2,4,6-TCP). Both X-ray crystal structures and isothermal titration calorimetry data indicated the association of two PCP molecules per PcpR, but only one 2,4,6-TCP molecule. The hydrophobic nature and hydrogen bonds of one binding cavity allowed the tight association of both PCP (Kd = 110 nM) and 2,4,6-TCP (Kd = 22.8 nM). However, the other cavity was unique to PCP with much weaker affinity (Kd = 70 µM) and thus its significance was not clear. Neither phenol nor benzoic acid displayed any significant affinity to PcpR, indicating a role of chlorine substitution in ligand specificity. When PcpR is compared with TcpR, a LysR-type regulator controlling the expression of 2,4,6-trichlorophenol degradation in Cupriavidus necator JMP134, most of the residues constituting the two inducer-binding cavities of PcpR are different, except for their general hydrophobic nature. The finding concurs that PcpR uses various polychlorophenols as long as it includes 2,4,6-trichlorophenol, as inducers; whereas TcpR is only responsive to 2,4,6-trichlorophenol.


Asunto(s)
Proteínas Bacterianas/química , Clorofenoles/metabolismo , Pentaclorofenol/metabolismo , Sphingomonadaceae/química , Sphingomonadaceae/metabolismo , Factores de Transcripción/química , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Factores de Transcripción/metabolismo
16.
J Biol Chem ; 287(14): 11592-601, 2012 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-22337878

RESUMEN

Calsequestrin, the major calcium storage protein in both cardiac and skeletal muscle, binds large amounts of Ca(2+) in the sarcoplasmic reticulum and releases them during muscle contraction. For the first time, the crystal structures of Ca(2+) complexes for both human (hCASQ1) and rabbit (rCASQ1) skeletal calsequestrin were determined, clearly defining their Ca(2+) sequestration capabilities through resolution of high- and low-affinity Ca(2+)-binding sites. rCASQ1 crystallized in low CaCl(2) buffer reveals three high-affinity Ca(2+) sites with trigonal bipyramidal, octahedral, and pentagonal bipyramidal coordination geometries, along with three low-affinity Ca(2+) sites. hCASQ1 crystallized in high CaCl(2) shows 15 Ca(2+) ions, including the six Ca(2+) ions in rCASQ1. Most of the low-affinity sites, some of which are µ-carboxylate-bridged, are established by the rotation of dimer interfaces, indicating cooperative Ca(2+) binding that is consistent with our atomic absorption spectroscopic data. On the basis of these findings, we propose a mechanism for the observed in vitro and in vivo dynamic high-capacity and low-affinity Ca(2+)-binding activity of calsequestrin.


Asunto(s)
Calcio/metabolismo , Calsecuestrina/metabolismo , Músculo Esquelético/metabolismo , Secuencia de Aminoácidos , Animales , Calsecuestrina/química , Cristalografía por Rayos X , Perros , Humanos , Luz , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Conejos , Dispersión de Radiación
17.
J Biol Chem ; 287(5): 3042-50, 2012 Jan 27.
Artículo en Inglés | MEDLINE | ID: mdl-22170046

RESUMEN

Calsequestrin (CASQ) serves as a major Ca(2+) storage/buffer protein in the sarcoplasmic reticulum (SR). When purified from skeletal muscle, CASQ1 is obtained in its glycosylated form. Here, we have confirmed the specific site and degree of glycosylation of native rabbit CASQ1 and have investigated its effect on critical properties of CASQ by comparison with the non-glycosylated recombinant form. Based on our comparative approach utilizing crystal structures, Ca(2+) binding capacities, analytical ultracentrifugation, and light-scattering profiles of the native and recombinant rabbit CASQ1, we propose a novel and dynamic role for glycosylation in CASQ. CASQ undergoes a unique degree of mannose trimming as it is trafficked from the proximal endoplasmic reticulum to the SR. The major glycoform of CASQ (GlcNAc(2)Man(9)) found in the proximal endoplasmic reticulum can severely hinder formation of the back-to-back interface, potentially preventing premature Ca(2+)-dependent polymerization of CASQ and ensuring its continuous mobility to the SR. Only trimmed glycans can stabilize both front-to-front and the back-to-back interfaces of CASQ through extensive hydrogen bonding and electrostatic interactions. Therefore, the mature glycoform of CASQ (GlcNAc(2)Man(1-4)) within the SR can be retained upon establishing a functional high capacity Ca(2+) binding polymer. In addition, based on the high resolution structures, we propose a molecular mechanism for the catecholaminergic polymorphic ventricular tachycardia (CPVT2) mutation, K206N.


Asunto(s)
Calcio/química , Calsecuestrina/química , Multimerización de Proteína/fisiología , Sustitución de Aminoácidos , Animales , Calcio/metabolismo , Calsecuestrina/genética , Calsecuestrina/metabolismo , Cristalografía por Rayos X , Retículo Endoplásmico/metabolismo , Glicosilación , Mutación Missense , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo
18.
Int J Mol Sci ; 13(11): 14326-43, 2012 Nov 06.
Artículo en Inglés | MEDLINE | ID: mdl-23203067

RESUMEN

Calsequestrin (CASQ) is a major Ca2+-storage/buffer protein present in the sarcoplasmic reticulum of both skeletal (CASQ1) and cardiac (CASQ2) muscles. CASQ has significant affinity for a number of pharmaceutical drugs with known muscular toxicities. Our approach, with in silico molecular docking, single crystal X-ray diffraction, and isothermal titration calorimetry (ITC), identified three distinct binding pockets on the surface of CASQ2, which overlap with 2-methyl-2,4-pentanediol (MPD) binding sites observed in the crystal structure. Those three receptor sites based on canine CASQ1 crystal structure gave a high correlation (R2 = 0.80) to our ITC data. Daunomycin, doxorubicin, thioridazine, and trifluoperazine showed strong affinity to the S1 site, which is a central cavity formed between three domains of CASQ2. Some of the moderate-affinity drugs and some high-affinity drugs like amlodipine and verapamil displayed their binding into S2 sites, which are the thioredoxin-like fold present in each CASQ domain. Docking predictions combined with dissociation constants imply that presence of large aromatic cores and less flexible functional groups determines the strength of binding affinity to CASQ. In addition, the predicted binding pockets for both caffeine and epigallocatechin overlapped with the S1 and S2 sites, suggesting competitive inhibition by these natural compounds as a plausible explanation for their antagonistic effects on cardiotoxic side effects.


Asunto(s)
Calsecuestrina/química , Preparaciones Farmacéuticas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Unión Competitiva , Calsecuestrina/metabolismo , Cinética , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Preparaciones Farmacéuticas/metabolismo , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Teoría Cuántica , Conejos
19.
Am J Obstet Gynecol ; 203(5): 494.e1-6, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20810099

RESUMEN

OBJECTIVE: This study determined the influence of a 2-component polyethylene glycol surgical sealant (Coseal) as an adhesion prevention device on sepsis-related mortality and/or systemic bacterial translocation to the spleen. STUDY DESIGN: A bacterial inoculum and telemetry probe were implanted in 50 treated and 49 untreated rats. Telemetry probes monitored core-body temperature to determine time of death. Spleens were collected on day 3 for quantitative bacteriology of Escherichia coli and Bacteroides fragilis. RESULTS: Median survival time and mortality of treated rats (37.0 hours, 54.0%) were noninferior to untreated rats (47.5 hours, 55.1%). Median E coli titers in treated rats (2.24 log colony forming units/spleen) were significantly less than untreated rats (4.32 log colony forming units/spleen). B fragilis titers were not different. CONCLUSION: This study demonstrates intraperitoneal administration of a 2-component polyethylene glycol surgical sealant as an adhesion prevention device does not alter time to death or sepsis-related mortality and/or systemic bacterial translocation to the spleen.


Asunto(s)
Infecciones por Bacteroides/prevención & control , Infecciones por Escherichia coli/prevención & control , Enfermedades Peritoneales/prevención & control , Polietilenglicoles/uso terapéutico , Bazo/microbiología , Animales , Infecciones por Bacteroides/microbiología , Bacteroides fragilis , Escherichia coli , Infecciones por Escherichia coli/microbiología , Femenino , Estimación de Kaplan-Meier , Enfermedades Peritoneales/microbiología , Ratas , Ratas Sprague-Dawley , Telemetría , Adherencias Tisulares/microbiología , Adherencias Tisulares/prevención & control
20.
J Invest Surg ; 32(5): 393-401, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-29420097

RESUMEN

Purpose/Aim: Powdered hemostats have been widely adopted for their ease-of-use; however, their efficacy has been limited resulting in applications restricted to low-level bleeds. This study investigates the use of bovine-derived gelatin particles (BGP) as a standalone hemostatic powder and compare BGP to commercially available microporous polysaccharide hemospheres (MPH). Materials and Methods: The powders were investigated for their hemostatic efficacy in a heparinized pre-clinical bleeding model limited to grade 1 and 2 bleeds on a validated intraoperative bleeding scale, which represents the accepted, clinical use of hemostatic powders. Results: At 10 minutes, the hemostatic success of lesions treated with BGP were 78% while MPH were 22%. The odds ratio for hemostatic success of BGP relative to MPH was 15.18 (95% CI: 7.37, 31.27). The 95% lower limit of the odds ratio was greater than 1. This indicates that BGP are superior to MPH (p < 0.001). The median time to hemostasis for BGP was 1.6 minutes and MPH was 14.5 minutes. The ratio for time to hemostasis of MPH relative to BGP was 9.23 (95% CI: 6.99, 12.19). This indicates that BGP achieve significantly faster time to hemostasis (p < 0.001). Conclusions: Characterization of tissue explant ultrastructure, particle size, and swelling revealed differences in the materials. BGP, in addition to absorbing fluid and concentrating clotting factors and platelets, integrate into the clot and stabilize the fibrin matrix. BGP have advantages over MPH in terms of speed and efficacy. BGP are a favorable biomaterial for further research that greatly improve the limited efficacy of powdered hemostats.


Asunto(s)
Materiales Biocompatibles/administración & dosificación , Pérdida de Sangre Quirúrgica/prevención & control , Gelatina/administración & dosificación , Hemostasis Quirúrgica/métodos , Polisacáridos/administración & dosificación , Animales , Materiales Biocompatibles/química , Modelos Animales de Enfermedad , Gelatina/química , Humanos , Hígado/cirugía , Masculino , Microscopía Electrónica de Rastreo , Modelos Animales , Tamaño de la Partícula , Polisacáridos/química , Polisacáridos/ultraestructura , Porosidad , Polvos , Sus scrofa
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