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1.
Nucleic Acids Res ; 49(17): 10120-10135, 2021 09 27.
Artículo en Inglés | MEDLINE | ID: mdl-34417623

RESUMEN

The iron-dependent regulator IdeR is the main transcriptional regulator controlling iron homeostasis genes in Actinobacteria, including species from the Corynebacterium, Mycobacterium and Streptomyces genera, as well as the erythromycin-producing bacterium Saccharopolyspora erythraea. Despite being a well-studied transcription factor since the identification of the Diphtheria toxin repressor DtxR three decades ago, the details of how IdeR proteins recognize their highly conserved 19-bp DNA target remain to be elucidated. IdeR makes few direct contacts with DNA bases in its target sequence, and we show here that these contacts are not required for target recognition. The results of our structural and mutational studies support a model wherein IdeR mainly uses an indirect readout mechanism, identifying its targets via the sequence-dependent DNA backbone structure rather than through specific contacts with the DNA bases. Furthermore, we show that IdeR efficiently recognizes a shorter palindromic sequence corresponding to a half binding site as compared to the full 19-bp target previously reported, expanding the number of potential target genes controlled by IdeR proteins.


Asunto(s)
Proteínas Bacterianas/metabolismo , Corynebacterium/genética , ADN Bacteriano/metabolismo , Mycobacterium/genética , Proteínas Represoras/metabolismo , Saccharopolyspora/genética , Streptomyces/genética , Proteínas Bacterianas/genética , Secuencia de Bases/genética , Sitios de Unión/genética , Corynebacterium/metabolismo , ADN Bacteriano/genética , Regulación Bacteriana de la Expresión Génica/genética , Hierro/química , Familia de Multigenes/genética , Mycobacterium/metabolismo , Proteínas Represoras/genética , Saccharopolyspora/metabolismo , Transducción de Señal/genética , Streptomyces/metabolismo , Transcripción Genética/genética
2.
Mol Genet Metab ; 136(3): 177-185, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35151535

RESUMEN

ß-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and catalyses the conversion of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid to ß-alanine and ß-aminoisobutyric acid, ammonia and CO2. To date, only a limited number of genetically confirmed patients with a complete ß-ureidopropionase deficiency have been reported. Here, we report on the clinical, biochemical and molecular findings of 10 newly identified ß-ureidopropionase deficient individuals. Patients presented mainly with neurological abnormalities and markedly elevated levels of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid in urine. Analysis of UPB1, encoding ß-ureidopropionase, showed 5 novel missense variants and two novel splice-site variants. Functional expression of the UPB1 variants in mammalian cells showed that recombinant ß-ureidopropionase carrying the p.Ala120Ser, p.Thr129Met, p.Ser300Leu and p.Asn345Ile variant yielded no or significantly decreased ß-ureidopropionase activity. Analysis of the crystal structure of human ß-ureidopropionase indicated that the point mutations affect substrate binding or prevent the proper subunit association to larger oligomers and thus a fully functional ß-ureidopropionase. A minigene approach showed that the intronic variants c.[364 + 6 T > G] and c.[916 + 1_916 + 2dup] led to skipping of exon 3 and 8, respectively, in the process of UPB1 pre-mRNA splicing. The c.[899C > T] (p.Ser300Leu) variant was identified in two unrelated Swedish ß-ureidopropionase patients, indicating that ß-ureidopropionase deficiency may be more common than anticipated.


Asunto(s)
Errores Innatos del Metabolismo de la Purina-Pirimidina , Precursores del ARN , Anomalías Múltiples , Amidohidrolasas/deficiencia , Amidohidrolasas/genética , Animales , Encefalopatías , Humanos , Mamíferos/genética , Trastornos del Movimiento , Mutación , Errores Innatos del Metabolismo de la Purina-Pirimidina/genética , beta-Alanina/genética , beta-Alanina/orina
3.
Biochem J ; 475(14): 2395-2416, 2018 07 31.
Artículo en Inglés | MEDLINE | ID: mdl-29976570

RESUMEN

ß-Ureidopropionase (ßUP) catalyzes the third step of the reductive pyrimidine catabolic pathway responsible for breakdown of uracil-, thymine- and pyrimidine-based antimetabolites such as 5-fluorouracil. Nitrilase-like ßUPs use a tetrad of conserved residues (Cys233, Lys196, Glu119 and Glu207) for catalysis and occur in a variety of oligomeric states. Positive co-operativity toward the substrate N-carbamoyl-ß-alanine and an oligomerization-dependent mechanism of substrate activation and product inhibition have been reported for the enzymes from some species but not others. Here, the activity of recombinant human ßUP is shown to be similarly regulated by substrate and product, but in a pH-dependent manner. Existing as a homodimer at pH 9, the enzyme increasingly associates to form octamers and larger oligomers with decreasing pH. Only at physiological pH is the enzyme responsive to effector binding, with N-carbamoyl-ß-alanine causing association to more active higher molecular mass species, and ß-alanine dissociation to inactive dimers. The parallel between the pH and ligand-induced effects suggests that protonation state changes play a crucial role in the allosteric regulation mechanism. Disruption of dimer-dimer interfaces by site-directed mutagenesis generated dimeric, inactive enzyme variants. The crystal structure of the T299C variant refined to 2.08 Šresolution revealed high structural conservation between human and fruit fly ßUP, and supports the hypothesis that enzyme activation by oligomer assembly involves ordering of loop regions forming the entrance to the active site at the dimer-dimer interface, effectively positioning the catalytically important Glu207 in the active site.


Asunto(s)
Amidohidrolasas/química , Multimerización de Proteína , Regulación Alostérica , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Sustitución de Aminoácidos , Animales , Antineoplásicos/farmacocinética , Cristalografía por Rayos X , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Humanos , Concentración de Iones de Hidrógeno , Mutación Missense , Dominios Proteicos
4.
Hum Mutat ; 39(7): 947-953, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29691939

RESUMEN

Dihydropyrimidine dehydrogenase (DPD) deficiency is associated with a variable clinical presentation. A family with three DPD-deficient patients presented with unusual clinical phenotypes including pregnancy-induced symptoms, transient visual impairment, severe developmental delay, cortical blindness, and delayed myelination in the brain. DPYD Sanger sequencing showed heterozygosity for the c.1905+1G>A mutation and a novel missense variant c.1700G>A (p.G567E). The recombinantly expressed p.G567E DPD variant showed increased temperature lability probably caused by structural rearrangements within the DPD protein. Genome sequencing of the affected son established compound heterozygosity for the c.1700G>A and an imperfect 115,731 bp inversion with breakpoints at chr1: 98,113,121 (intron 8) and chr1: 97,997,390 (intron 12) of the DPYD associated with a 4 bp deletion (chr1: 97,997,386_97,997,389del). Whole exome and mitochondrial DNA analyses for the mother and daughter did not reveal additional mutated genes of significance. Thus, an inversion in DPYD should be considered in patients with an inconclusive genotype or unusual clinical phenotype.


Asunto(s)
Deficiencia de Dihidropirimidina Deshidrogenasa/genética , Dihidrouracilo Deshidrogenasa (NADP)/genética , Mutación Missense/genética , Paraparesia Espástica/genética , Adolescente , Adulto , Preescolar , Mapeo Cromosómico , Deficiencia de Dihidropirimidina Deshidrogenasa/fisiopatología , Exones/genética , Femenino , Genotipo , Heterocigoto , Humanos , Lactante , Intrones/genética , Masculino , Paraparesia Espástica/fisiopatología , Fenotipo , Eliminación de Secuencia/genética , Secuenciación Completa del Genoma
6.
Biochim Biophys Acta Mol Basis Dis ; 1863(3): 721-730, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-28024938

RESUMEN

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). Genetic variations in DPD have emerged as predictive risk factors for severe fluoropyrimidine toxicity. Here, we report novel and rare genetic variants underlying DPD deficiency in 9 cancer patients presenting with severe fluoropyrimidine-associated toxicity. All patients possessed a strongly reduced DPD activity, ranging from 9 to 53% of controls. Analysis of the DPD gene (DPYD) showed the presence of 21 variable sites including 4 novel and 4 very rare aberrations: 3 missense mutations, 2 splice-site mutations, 1 intronic mutation, a deletion of 21 nucleotides and a genomic amplification of exons 9-12. Two novel/rare variants (c.2843T>C, c.321+1G>A) were present in multiple, unrelated patients. Functional analysis of recombinantly-expressed DPD mutants carrying the p.I948T and p.G284V mutation showed residual DPD activities of 30% and 0.5%, respectively. Analysis of a DPD homology model indicated that the p.I948T and p.G284V mutations may affect electron transfer and the binding of FAD, respectively. cDNA analysis showed that the c.321+1G>A mutation in DPYD leads to skipping of exon 4 immediately upstream of the mutated splice-donor site in the process of DPD pre-mRNA splicing. A lethal toxicity in two DPD patients suggests that fluoropyrimidines combined with other therapies such as radiotherapy might be particularly toxic for DPD deficient patients. Our study advocates a more comprehensive genotyping approach combined with phenotyping strategies for upfront screening for DPD deficiency to ensure the safe administration of fluoropyrimidines.


Asunto(s)
Antimetabolitos Antineoplásicos/efectos adversos , Capecitabina/efectos adversos , Dihidrouracilo Deshidrogenasa (NADP)/genética , Fluorouracilo/efectos adversos , Mutación , Empalme del ARN , Anciano , Deficiencia de Dihidropirimidina Deshidrogenasa/complicaciones , Deficiencia de Dihidropirimidina Deshidrogenasa/genética , Femenino , Amplificación de Genes , Células HEK293 , Humanos , Masculino , Persona de Mediana Edad , Modelos Moleculares , Mutación Missense , Neoplasias/complicaciones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Variantes Farmacogenómicas , Eliminación de Secuencia
7.
FEBS J ; 284(22): 3895-3914, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28963762

RESUMEN

Alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 is a promising biocatalyst for redox transformations of arylsubstituted sec-alcohols and ketones. The enzyme is stereoselective in the oxidation of 1-phenylethanol with a 300-fold preference for the (S)-enantiomer. The low catalytic efficiency with (R)-1-phenylethanol has been attributed to nonproductive binding of this substrate at the active site. Aiming to modify the enantioselectivity, to rather favor the (R)-alcohol, and also test the possible involvement of nonproductive substrate binding as a mechanism in substrate discrimination, we performed directed laboratory evolution of ADH-A. Three targeted sites that contribute to the active-site cavity were exposed to saturation mutagenesis in a stepwise manner and the generated variants were selected for improved catalytic activity with (R)-1-phenylethanol. After three subsequent rounds of mutagenesis, selection and structure-function analysis of isolated ADH-A variants, we conclude: (a) W295 has a key role as a structural determinant in the discrimination between (R)- and (S)-1-phenylethanol and a W295A substitution fundamentally changes the stereoselectivity of the protein. One observable effect is a faster rate of NADH release, which changes the rate-limiting step of the catalytic cycle from coenzyme release to hydride transfer. (b) The obtained change in enantiopreference, from the (S)- to the (R)-alcohol, can be partly explained by a shift in the nonproductive substrate-binding modes. DATABASE: Structural data are available in the Protein Data Bank with accession codes 5o8q for A2, 5o8h for A2C2, 5o9f for A2C3, and 5o9d for A2C2B1.


Asunto(s)
Alcohol Deshidrogenasa/química , Alcohol Deshidrogenasa/metabolismo , Coenzimas/metabolismo , Alcohol Feniletílico/metabolismo , Rhodococcus/enzimología , Alcohol Deshidrogenasa/genética , Sitios de Unión , Catálisis , Dominio Catalítico , Cinética , Mutagénesis Sitio-Dirigida , Mutación/genética , Oxidación-Reducción , Conformación Proteica , Estereoisomerismo , Especificidad por Sustrato
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