RESUMEN
The eukaryotic microsomal cytochrome P450 systems consist of a cytochrome P450 enzyme (P450) and a cytochrome P450 redox partner, which generally is a cytochrome P450 reductase (CPR) that supplies electrons from NADPH. However, alternative electron donors may exist such as cytochrome b5 reductase and cytochrome b5 (CBR and CYB5, respectively) via, which is NADH-dependent and are also anchored to the endoplasmic reticulum. In the carotenogenic yeast Xanthophyllomyces dendrorhous, three P450-encoding genes have been described: crtS is involved in carotenogenesis and the CYP51 and CYP61 genes are both implicated in ergosterol biosynthesis. This yeast has a single CPR (encoded by the crtR gene), and a crtR- mutant does not produce astaxanthin. Considering that this mutant is viable, the existence of alternative cytochrome P450 electron donors like CBR and CYB5 could operate in this yeast. The aim of this work was to characterize the X. dendrorhous CBR encoding gene and to study its involvement in P450 reactions in ergosterol and carotenoid biosynthesis. Two CBRs genes were identified (CBR.1 and CBR.2), and deletion mutants were constructed. The two mutants and the wild-type strain showed similar sterol production, with ergosterol being the main sterol produced. The crtR- mutant strain produced a lower proportion of ergosterol than did the parental strain. These results indicate that even though one of the two CBR genes could be involved in ergosterol biosynthesis, crtR complements their absence in the cbr- mutant strains, at least for ergosterol production. The higher NADH-dependent cytochrome c reductase activity together with the higher transcript levels of CBR.1 and CYB5 in the crtR- mutant as well as the lower NADH-dependent activity in CBS-cbr.1- strongly suggest that CBR.1-CYB5 via participates as an alternative electron donor pathway for P450 enzymes involved in ergosterol biosynthesis in X. dendrorhous.
Asunto(s)
Basidiomycota/genética , Citocromo-B(5) Reductasa/genética , Proteínas Fúngicas/genética , Secuencia de Aminoácidos , Basidiomycota/enzimología , Carotenoides/biosíntesis , Carotenoides/genética , Citocromo-B(5) Reductasa/química , Citocromo-B(5) Reductasa/metabolismo , Ergosterol/biosíntesis , Ergosterol/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Datos de Secuencia MolecularRESUMEN
Congenital methemoglobinemia due to NADH-cytochrome b5 reductase 3 (CYB5R3) deficiency is an autosomal recessive disorder that occurs sporadically worldwide, although endemic clusters of this disorder have been identified in certain ethnic groups. It is present as two distinct phenotypes, type I and type II. Type I methemoglobinemia is characterized by CYB5R3 enzyme deficiency restricted to erythrocytes and is associated with benign cyanosis. The less frequent type II methemoglobinemia is associated with generalized CYB5R3 deficiency affecting all cells and is lethal in early infancy. Here we describe the molecular basis of type I methemoglobinemia due to CYB5R3 deficiency in four patients from three distinct ethnic backgrounds, Asian Indian, Mexican and Greek. The CYB5R3 gene of three probands with type I methemoglobinemia and their relatives were sequenced revealing several putative causative mutations; in one subject multiple mutations were present. Two novel mutations, S54R and F157C, were identified and the previously described A179T, V253M mutations were also identified. All these point mutations mapped to the NADH binding domain and or the FAD binding domain. Each has the potential to sterically hinder cofactor binding causing instability of the CYB5R3 protein. Wild-type CYB5R3, as well as two of these novel mutations, S54R and F157C, was amplified, cloned, and purified recombinant peptide obtained. Kinetic and thermodynamic studies of these proteins show that the above mutations lead to decreased thermal stability.
Asunto(s)
Citocromo-B(5) Reductasa/genética , Metahemoglobinemia/etnología , Metahemoglobinemia/genética , Mutación , Sitios de Unión/genética , Citocromo-B(5) Reductasa/química , Estabilidad de Enzimas/genética , Flavina-Adenina Dinucleótido/metabolismo , Grecia , Humanos , India , Cinética , México , NAD/metabolismo , Fenotipo , TermodinámicaRESUMEN
Type II methemoglobinemia is a somatic deficiency of cytochrome b5 reductase with severe global neurologic impairment. We report a novel mutation in exon 3 of the CYB5R3 gene on chromosome 22 consisting of homozygous 1-base pair (bp) deletion noted as c.215delG; p.Gly72AlafsX100. The patient had improvement of gross motor skills, chewing, and swallowing that may be due to the initiation of daily ascorbic acid therapy. We hypothesize that a possible response to ascorbic acid may be related to the effect of making additional ferrous iron available for its role as a cofactor in carnitine synthesis.
Asunto(s)
Citocromo-B(5) Reductasa/genética , Metahemoglobinemia/genética , Eliminación de Secuencia , Adulto , Ácido Ascórbico/uso terapéutico , Niño , Discinesias/tratamiento farmacológico , Discinesias/etiología , Discinesias/genética , Femenino , Honduras , Humanos , Masculino , Metahemoglobinemia/complicaciones , Metahemoglobinemia/tratamiento farmacológico , Madres , Análisis de Secuencia de ADN , Resultado del Tratamiento , Vitaminas/uso terapéuticoRESUMEN
The present work aims to study a new NADH-cytochrome b5 reductase (cb5r) from Mucor racemosus PTCC 5305. A cDNA coding for cb5r was isolated from a Mucor racemosus PTCC 5305 cDNA library. The nucleotide sequence of the cDNA including coding and sequences flanking regions was determined. The open reading frame starting from ATG and ending with TAG stop codon encoded 228 amino acids and displayed the closest similarity (73%) with Mortierella alpina cb5r. Lack of hydrophobic residues in the N-terminal sequence was apparent, suggesting that the enzyme is a soluble isoform. The coding sequence was then cloned in the pET16b transcription vector carrying an N-terminal-linked His-Tag sequence and expressed in Escherichia coli BL21 (DE3). The enzyme was then homogeneously purified by a metal affinity column. The recombinant Mucor enzyme was shown to have its optimal activity at pH and temperature of about 7.5 and 40 degrees C, respectively. The apparent K(m) value was calculated to be 13 microM for ferricyanide. To our knowledge, this is the first report on cloning and expression of a native fungal soluble isoform of NADH-cytochrome b5 reductase in E. coli.
Asunto(s)
Citocromo-B(5) Reductasa/genética , ADN Complementario/genética , Escherichia coli/genética , Vectores Genéticos/genética , Mucor/enzimología , Secuencia de Bases , Clonación Molecular , Citocromo-B(5) Reductasa/metabolismo , Biblioteca de Genes , Isoenzimas/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Transcripción GenéticaRESUMEN
NADH-methaemoglobin reductase deficiency has been found in 4 Cuban families; 3 subjects carried the mild form of the deficiency while in 2 sibs of the fourth family the deficiency was associated with neurological involvement. The parents in this family were consanguinous and the sibs were shown to be homozygous for a new fast electrophoretic variant. It was named Diaphorase Santiago de Cuba.