Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
Más filtros










Base de datos
Intervalo de año de publicación
1.
J Biol Chem ; 293(17): 6374-6386, 2018 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-29496995

RESUMEN

Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques.


Asunto(s)
Cianatos , Cianuros , Peroxidasa , Placa Aterosclerótica/enzimología , Carbamilación de Proteína , Animales , Citrulina/análogos & derivados , Citrulina/química , Citrulina/genética , Citrulina/metabolismo , Cianatos/química , Cianatos/metabolismo , Cianuros/química , Cianuros/metabolismo , Humanos , Ratones , Ratones Noqueados , Oxidación-Reducción , Peroxidasa/química , Peroxidasa/genética , Peroxidasa/metabolismo , Placa Aterosclerótica/genética , Placa Aterosclerótica/patología
2.
J Biol Chem ; 292(20): 8244-8261, 2017 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-28348079

RESUMEN

Myeloperoxidase (MPO) is synthesized by neutrophil and monocyte precursor cells and contributes to host defense by mediating microbial killing. Although several steps in MPO biosynthesis and processing have been elucidated, many questions remained, such as the structure-function relationship of monomeric unprocessed proMPO versus the mature dimeric MPO and the functional role of the propeptide. Here we have presented the first and high resolution (at 1.25 Å) crystal structure of proMPO and its solution structure obtained by small-angle X-ray scattering. Promyeloperoxidase hosts five occupied glycosylation sites and six intrachain cystine bridges with Cys-158 of the very flexible N-terminal propeptide being covalently linked to Cys-319 and thereby hindering homodimerization. Furthermore, the structure revealed (i) the binding site of proMPO-processing proconvertase, (ii) the structural motif for subsequent cleavage to the heavy and light chains of mature MPO protomers, and (iii) three covalent bonds between heme and the protein. Studies of the mutants C158A, C319A, and C158A/C319A demonstrated significant differences from the wild-type protein, including diminished enzymatic activity and prevention of export to the Golgi due to prolonged association with the chaperone calnexin. These structural and functional findings provide novel insights into MPO biosynthesis and processing.


Asunto(s)
Precursores Enzimáticos , Peroxidasa , Sustitución de Aminoácidos , Calnexina/química , Calnexina/genética , Calnexina/metabolismo , Cristalografía por Rayos X , Activación Enzimática/fisiología , Precursores Enzimáticos/biosíntesis , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Aparato de Golgi/enzimología , Aparato de Golgi/genética , Células HEK293 , Humanos , Células K562 , Mutación Missense , Peroxidasa/biosíntesis , Peroxidasa/química , Peroxidasa/genética , Dominios Proteicos
3.
J Biol Chem ; 292(11): 4583-4592, 2017 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-28154175

RESUMEN

Human peroxidasin 1 is a homotrimeric multidomain peroxidase that is secreted to the extracellular matrix. The heme enzyme was shown to release hypobromous acid that mediates the formation of specific covalent sulfilimine bonds to reinforce collagen IV in basement membranes. Maturation by proteolytic cleavage is known to activate the enzyme. Here, we present the first multimixing stopped-flow study on a fully functional truncated variant of human peroxidasin 1 comprising four immunoglobulin-like domains and the catalytically active peroxidase domain. The kinetic data unravel the so far unknown substrate specificity and mechanism of halide oxidation of human peroxidasin 1. The heme enzyme is shown to follow the halogenation cycle that is induced by the rapid H2O2-mediated oxidation of the ferric enzyme to the redox intermediate compound I. We demonstrate that chloride cannot act as a two-electron donor of compound I, whereas thiocyanate, iodide, and bromide efficiently restore the ferric resting state. We present all relevant apparent bimolecular rate constants, the spectral signatures of the redox intermediates, and the standard reduction potential of the Fe(III)/Fe(II) couple, and we demonstrate that the prosthetic heme group is post-translationally modified and cross-linked with the protein. These structural features provide the basis of human peroxidasin 1 to act as an effective generator of hypobromous acid, which mediates the formation of covalent cross-links in collagen IV.


Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Peroxidasa/metabolismo , Bromuros/metabolismo , Dominio Catalítico , Cloruros/metabolismo , Colágeno Tipo IV/metabolismo , Proteínas de la Matriz Extracelular/química , Compuestos Férricos/metabolismo , Halogenación , Humanos , Peróxido de Hidrógeno/metabolismo , Yoduros/metabolismo , Cinética , Oxidación-Reducción , Peroxidasa/química , Dominios Proteicos , Especificidad por Sustrato , Tiocianatos/metabolismo , Peroxidasina
4.
J Biol Chem ; 290(17): 10876-90, 2015 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-25713063

RESUMEN

Human peroxidasin 1 (hsPxd01) is a multidomain heme peroxidase that uses bromide as a cofactor for the formation of sulfilimine cross-links. The latter confers critical structural reinforcement to collagen IV scaffolds. Here, hsPxd01 and various truncated variants lacking nonenzymatic domains were recombinantly expressed in HEK cell lines. The N-glycosylation site occupancy and disulfide pattern, the oligomeric structure, and unfolding pathway are reported. The homotrimeric iron protein contains a covalently bound ferric high spin heme per subunit with a standard reduction potential of the Fe(III)/Fe(II) couple of -233 ± 5 mV at pH 7.0. Despite sequence homology at the active site and biophysical properties similar to human peroxidases, the catalytic efficiency of bromide oxidation (kcat/KM(app)) of full-length hsPxd01 is rather low but increased upon truncation. This is discussed with respect to its structure and proposed biosynthetic function in collagen IV cross-linking.


Asunto(s)
Antígenos de Neoplasias/química , Colágeno Tipo IV/química , Hierro/química , Receptores de Interleucina-1/química , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Catálisis , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Glicosilación , Células HEK293 , Humanos , Hierro/metabolismo , Oxidación-Reducción , Peroxidasas , Estructura Terciaria de Proteína , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Relación Estructura-Actividad
5.
Arch Biochem Biophys ; 574: 108-19, 2015 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-25575902

RESUMEN

Four heme peroxidase superfamilies (peroxidase-catalase, peroxidase-cyclooxygenase, peroxidase-chlorite dismutase and peroxidase-peroxygenase superfamily) arose independently during evolution, which differ in overall fold, active site architecture and enzymatic activities. The redox cofactor is heme b or posttranslationally modified heme that is ligated by either histidine or cysteine. Heme peroxidases are found in all kingdoms of life and typically catalyze the one- and two-electron oxidation of a myriad of organic and inorganic substrates. In addition to this peroxidatic activity distinct (sub)families show pronounced catalase, cyclooxygenase, chlorite dismutase or peroxygenase activities. Here we describe the phylogeny of these four superfamilies and present the most important sequence signatures and active site architectures. The classification of families is described as well as important turning points in evolution. We show that at least three heme peroxidase superfamilies have ancient prokaryotic roots with several alternative ways of divergent evolution. In later evolutionary steps, they almost always produced highly evolved and specialized clades of peroxidases in eukaryotic kingdoms with a significant portion of such genes involved in coding various fusion proteins with novel physiological functions.


Asunto(s)
Evolución Biológica , Peroxidasas/metabolismo , Catalasa/metabolismo , Hemo , Modelos Moleculares , Peroxidasas/química , Prostaglandina-Endoperóxido Sintasas/metabolismo , Conformación Proteica
6.
J Biol Chem ; 288(38): 27181-27199, 2013 Sep 20.
Artículo en Inglés | MEDLINE | ID: mdl-23918925

RESUMEN

Reconstructing the phylogenetic relationships of the main evolutionary lines of the mammalian peroxidases lactoperoxidase and myeloperoxidase revealed the presence of novel bacterial heme peroxidase subfamilies. Here, for the first time, an ancestral bacterial heme peroxidase is shown to possess a very high bromide oxidation activity (besides conventional peroxidase activity). The recombinant protein allowed monitoring of the autocatalytic peroxide-driven formation of covalent heme to protein bonds. Thereby, the high spin ferric rhombic heme spectrum became similar to lactoperoxidase, the standard reduction potential of the Fe(III)/Fe(II) couple shifted to more positive values (-145 ± 10 mV at pH 7), and the conformational and thermal stability of the protein increased significantly. We discuss structure-function relationships of this new peroxidase in relation to its mammalian counterparts and ask for its putative physiological role.


Asunto(s)
Proteínas Bacterianas/química , Bromuros/química , Cianobacterias/enzimología , Hemo/química , Peroxidasa/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bromuros/metabolismo , Cianobacterias/genética , Estabilidad de Enzimas/fisiología , Hemo/genética , Hemo/metabolismo , Concentración de Iones de Hidrógeno , Oxidación-Reducción , Peroxidasa/genética , Peroxidasa/metabolismo
7.
Chem Biodivers ; 9(9): 1776-93, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22976969

RESUMEN

Peroxidasins represent the subfamily 2 of the peroxidase-cyclooxygenase superfamily and are closely related to chordata peroxidases (subfamily 1) and peroxinectins (subfamily 3). They are multidomain proteins containing a heme peroxidase domain with high homology to human lactoperoxidase that mediates one- and two-electron oxidation reactions. Additional domains of the secreted and glycosylated metalloproteins are type C-like immunoglobulin domains, typical leucine-rich repeats, as well as a von Willebrand factor C module. These are typical motifs of extracellular proteins that mediate protein-protein interactions. We have reconstructed the phylogeny of this new family of oxidoreductases and show the presence of four invertebrate clades as well as one vertebrate clade that includes also two different human representatives. The variability of domain assembly in the various clades was analyzed, as was the occurrence of relevant catalytic residues in the peroxidase domain based on the knowledge of catalysis of the mammalian homologues. Finally, the few reports on expression, localization, enzymatic activity, and physiological roles in the model organisms Drosophila melanogaster, Caenorhabditis elegans, and Homo sapiens are critically reviewed. Roles attributed to peroxidasins include antimicrobial defense, extracellular matrix formation, and consolidation at various developmental stages. Many research questions need to be solved in future, including detailed biochemical/physical studies and elucidation of the three dimensional structure of a model peroxidasin as well as the relation and interplay of the domains and the in vivo functions in various organisms including man.


Asunto(s)
Evolución Molecular , Secuencia de Aminoácidos , Animales , Cordados , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Peroxidasa/química , Peroxidasa/genética , Peroxidasa/metabolismo , Peroxidasas/química , Peroxidasas/genética , Filogenia , Alineación de Secuencia , Peroxidasina
8.
Arch Biochem Biophys ; 516(1): 21-8, 2011 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-21967851

RESUMEN

In mammalian peroxidases the proximal histidine is in close interaction with a fully conserved asparagine which in turn is hydrogen bonded with an arginine that stabilizes the propionate substituent of pyrrol ring D in bent conformation. In order to probe the role of this rigid proximal architecture for structural integrity and catalysis of human myeloperoxidase (MPO), the variants Asn421Asp, Arg333Ala and Arg333Lys have been recombinantly expressed in HEK cell lines. The standard reduction potential of the Fe(III)/Fe(II) couple of Asn421Asp was still wild-type-like (-50mV at pH 7.0) but the spectral properties of the ferric and ferrous forms as well as of higher oxidation states showed significant differences. Additionally, rates of ligand binding and oxidation of both one- and two-electron donors were diminished. The effect of exchange of Arg333 was even more dramatic. We did not succeed in production of mutant proteins that could bind heme at the active site. The importance of this His-Asn-Arg triad in linking the heme iron with the propionate at pyrrol ring D for heme insertion and binding as well as in maintenance of the architecture of the substrate binding site(s) at the entrance to the heme cavity is discussed.


Asunto(s)
Hemo/metabolismo , Peroxidasa/química , Peroxidasa/metabolismo , Sustitución de Aminoácidos , Arginina/química , Arginina/genética , Arginina/metabolismo , Asparagina/química , Asparagina/genética , Asparagina/metabolismo , Sitios de Unión , Línea Celular , Clonación Molecular , Compuestos Férricos/metabolismo , Compuestos Ferrosos/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Humanos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxidación-Reducción , Peroxidasa/genética
9.
Biochemistry ; 50(37): 7987-94, 2011 Sep 20.
Artículo en Inglés | MEDLINE | ID: mdl-21854003

RESUMEN

Myeloperoxidase (MPO) is the most abundant neutrophil enzyme and catalyzes predominantly the two-electron oxidation of ubiquitous chloride to generate the potent bleaching hypochlorous acid, thus contributing to pathogen killing as well as inflammatory diseases. Its catalytic properties are closely related with unique posttranslational modifications of its prosthetic group. In MPO, modified heme b is covalently bound to the protein via two ester linkages and one sulfonium ion linkage with a strong impact on its (electronic) structure and biophysical and chemical properties. Here, the thermodynamics of the one-electron reduction of the ferric heme in wild-type recombinant MPO and variants with disrupted heme-protein bonds (M243V, E242Q, and D94V) have been investigated by thin-layer spectroelectrochemistry. It turns out that neither the oligomeric structure nor the N-terminal extension in recombinant MPO modifies the peculiar positive reduction potential (E°' = 0.001 V at 25 °C and pH 7.0) or the enthalpy or entropy of the Fe(III) to Fe(II) reduction. By contrast, upon disruption of the MPO-typical sulfonium ion linkage, the reduction potential is significantly lower (-0.182 V). The M243V mutant has an enthalpically stabilized ferric state, whereas its ferrous form is entropically favored because of the loss of rigidity of the distal H-bonding network. Exchange of an adjacent ester bond (E242Q) induced similar but less pronounced effects (E°' = -0.094 V), whereas in the D94V variant (E°' = -0.060 V), formation of the ferrous state is entropically disfavored. These findings are discussed with respect to the chlorination and bromination activity of the wild-type protein and the mutants.


Asunto(s)
Hemoproteínas/metabolismo , Peroxidasa/metabolismo , Termodinámica , Animales , Células CHO , Cricetinae , Cricetulus , Hemoproteínas/química , Humanos , Oxidación-Reducción , Peroxidasa/química , Unión Proteica/fisiología , Estructura Secundaria de Proteína
10.
J Biol Chem ; 284(38): 25929-37, 2009 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-19608745

RESUMEN

In heme enzymes belonging to the peroxidase-cyclooxygenase superfamily the proximal histidine is in close interaction with a fully conserved asparagine. The crystal structure of a mixture of glycoforms of myeloperoxidase (MPO) purified from granules of human leukocytes prompted us to revise the orientation of this asparagine and the protonation status of the proximal histidine. The data we present contrast with previous MPO structures, but are strongly supported by molecular dynamics simulations. Moreover, comprehensive analysis of published lactoperoxidase structures suggest that the described proximal heme architecture is a general structural feature of animal heme peroxidases. Its importance is underlined by the fact that the MPO variant N421D, recombinantly expressed in mammalian cell lines, exhibited modified spectral properties and diminished catalytic activity compared with wild-type recombinant MPO. It completely lost its ability to oxidize chloride to hypochlorous acid, which is a characteristic feature of MPO and essential for its role in host defense. The presented crystal structure of MPO revealed further important differences compared with the published structures including the extent of glycosylation, interaction between light and heavy polypeptides, as well as heme to protein covalent bonds. These data are discussed with respect to biosynthesis and post-translational maturation of MPO as well as to its peculiar biochemical and biophysical properties.


Asunto(s)
Asparagina/química , Histidina/química , Leucocitos/enzimología , Peroxidasa/química , Asparagina/genética , Asparagina/metabolismo , Línea Celular , Cloruros/metabolismo , Cristalografía por Rayos X , Glicosilación , Hemo/química , Hemo/genética , Hemo/metabolismo , Histidina/genética , Histidina/metabolismo , Humanos , Ácido Hipocloroso/metabolismo , Lactoperoxidasa/química , Lactoperoxidasa/genética , Lactoperoxidasa/metabolismo , Mutación Missense , Oxidación-Reducción , Peroxidasa/genética , Peroxidasa/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Estructura Terciaria de Proteína/fisiología
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA