RESUMEN
CytC3, a member of the recently discovered class of nonheme Fe(II) and alpha-ketoglutarate (alphaKG)-dependent halogenases, catalyzes the double chlorination of L-2-aminobutyric acid (Aba) to produce a known Streptomyces antibiotic, gamma,gamma-dichloroaminobutyrate. Unlike the majority of the Fe(II)-alphaKG-dependent enzymes that catalyze hydroxylation reactions, halogenases catalyze a transfer of halides. To examine the important enzymatic features that discriminate between chlorination and hydroxylation, the crystal structures of CytC3 both with and without alphaKG/Fe(II) have been solved to 2.2 A resolution. These structures capture CytC3 in an open active site conformation, in which no chloride is bound to iron. Comparison of the open conformation of CytC3 with the closed conformation of another nonheme iron halogenase, SyrB2, suggests two important criteria for creating an enzyme-bound Fe-Cl catalyst: (1) the presence of a hydrogen-bonding network between the chloride and surrounding residues, and (2) the presence of a hydrophobic pocket in which the chloride resides.
Asunto(s)
Proteínas Bacterianas/química , Dominio Catalítico , Hierro/química , Ácidos Cetoglutáricos/química , Proteínas de Hierro no Heme/química , Streptomyces/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Cloro/química , Cloro/metabolismo , Cristalografía por Rayos X , Halogenación , Hidroxilación , Hierro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Datos de Secuencia Molecular , Proteínas de Hierro no Heme/genética , Proteínas de Hierro no Heme/aislamiento & purificación , Proteínas de Hierro no Heme/metabolismo , Conformación Proteica , Alineación de SecuenciaRESUMEN
AIM: To prepare and characterize antibody against Memo protein and to detect the tissue distribution of Memo in mice. METHODS: Fusion protein GST-Memo was expressed and purified, and polyclonal antibody against Memo was prepared by immunizing mice. A FLAG-tagged eukaryotic expression vector pcDNA3-FLAG-Memo was constructed. The specificity of the antibody was detected by Western blot. RESULTS: An eukaryotic expression vector pcDNA3-FLAG-Memo was obtained. The polyclonal antibody was found to be specific to Memo. Memo protein was widely expressed in mouse tissues using the obtained antibody in Western blot. CONCLUSION: Antibody specific to Memo has been successfully obtained, which provides useful tool for investigation into Memo-associated mechanisms of tumor metastasis and invasiveness.
Asunto(s)
Anticuerpos/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Proteínas de Hierro no Heme/inmunología , Proteínas de Hierro no Heme/metabolismo , Animales , Western Blotting , Línea Celular , Escherichia coli/genética , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Ratones , Proteínas de Hierro no Heme/biosíntesis , Proteínas de Hierro no Heme/aislamiento & purificación , Especificidad de Órganos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoAsunto(s)
Amianto/efectos adversos , Enfermedades Pulmonares/etiología , Enfermedades Pleurales/etiología , Contaminantes Atmosféricos/efectos adversos , Contaminantes Ocupacionales del Aire/efectos adversos , Amianto/química , Amianto/aislamiento & purificación , Asbestosis/epidemiología , Líquido del Lavado Bronquioalveolar/química , Carcinógenos Ambientales/efectos adversos , Humanos , Pulmón/química , Pulmón/ultraestructura , Enfermedades Pulmonares/epidemiología , Enfermedades Pulmonares/prevención & control , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/prevención & control , Mesotelioma/etiología , Mesotelioma/prevención & control , Fibras Minerales/análisis , Proteínas de Hierro no Heme/aislamiento & purificación , Enfermedades Pleurales/epidemiología , Enfermedades Pleurales/prevención & control , Neoplasias Pleurales/etiología , Neoplasias Pleurales/prevención & controlAsunto(s)
Corteza Cerebral/enzimología , Sistema Enzimático del Citocromo P-450/metabolismo , NADH Deshidrogenasa/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Proteínas de Hierro no Heme/metabolismo , Retina/metabolismo , Animales , Animales Recién Nacidos , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Masculino , Microsomas/enzimología , Microsomas Hepáticos/enzimología , NADPH-Ferrihemoproteína Reductasa , Proteínas de Hierro no Heme/química , Proteínas de Hierro no Heme/aislamiento & purificación , Oxidación-Reducción , Ratas , Ratas Wistar , Retina/crecimiento & desarrolloRESUMEN
A new component, which substitutes cytochrome P-450 as an acceptor of reducing equivalents from NADPH-cytochrome P-450 reductase, was identified in the bovine retina microsomal monooxigenase system, which does not contain cytochrome P-450. This component is a non-heme iron-containing protein with molecular mass of 66 kDa. The properties of the protein from the bovine retina are similar to those of MIP, a non-heme iron-containing protein from the heart microsomal monooxigenase system, in which cytochrome P-450 was not identified, either. Activation of the microsomal monooxigenase system (an increase in the NADPH-cytochrome P-450 reductase activity, an increased rate of microsomal NADPH oxidation) was shown in the retina upon long-term intensive illumination. It was shown also that the development of hereditary degeneration of the retina in rats was accompanied by activation of the specific microsomal monooxigenase system in the target tissues (retina, brain cortex) irrespective of its composition (cytochrome P-450 or non-heme iron-containing protein).