RESUMO
The acyl-CoA dehydrogenases (ACDs) are a family of related enzymes which catalyze the alpha,beta-dehydrogenation of acyl-CoA esters, transferring electrons to electron transferring flavoprotein. We have recently cloned and characterized the cDNA for human short/branched chain acyl-CoA dehydrogenase (SBCAD). Based on homology with the other ACDs we hypothesized that E381 is the catalytic residue for this enzyme. Alteration of this amino acid to glutamine, glycine or arginine resulted in an inactive enzyme. Substitution of aspartate at this position led to an enzyme with reduced activity compared to the wild type. An E381G/G260E double mutation (which places a glutamate in a position homologous to the catalytic residue identified in other members of this gene family) restored enzyme activity. These data confirm the crucial nature of E381 to the activity of this enzyme and strongly support its role as the alpha-proton abstracting base.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Oxirredutases/química , Oxirredutases/metabolismo , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação Puntual , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Dimethylglycine dehydrogenase (DMGDH; E.C. 1.5.99.2) is an enzyme involved in the catabolism of choline, catalyzing the oxidative demethylation of dimethylglycine (DMG) to form sarcosine. Subsequently, sarcosine dehydrogenase (SDH; E.C. 1.5.99.1) converts sarcosine to glycine via a similar reaction. Both enzymes are found as monomers in the mitochondrial matrix, and both contain 1 mol of covalently bound flavin adenine dinucleotide. DMGDH and SDH also utilize a noncovalently bound folate coenzyme that receives the "1-carbon" groups that are removed by DMGDH and SDH, forming "active formaldehyde." We have recently described a new inborn error of metabolism of DMGDH characterized by an unusual fish-like body odor. To augment our study of this new disorder, we have isolated two human genomic clones that together contain 16 exons of coding sequence for the hDMGDH gene. Fluorescent in situ hybridization analysis of the hDMGDH gene indicates that it is found on chromosome 5q12.2-q12.3. In addition, several polymorphisms have been identified in the hDMGDH cDNA sequence. Population analysis of two Ser/Pro polymorphisms found 367 amino acids apart reveals a skew of alleles, with the haplotypes Ser/Pro or Pro/Ser (79%) overrepresented compared to the number of Ser/Ser or Pro/Pro alleles observed. Possible functional consequences of these findings are discussed. Characterization of the gene structure for hDMGDH will aid in the study of patients with inherited defects of this enzyme.
Assuntos
Genes/genética , Oxirredutases N-Desmetilantes/genética , Alelos , Sequência de Aminoácidos , Mapeamento Cromossômico , Cromossomos Humanos Par 5/genética , Clonagem Molecular , DNA/química , DNA/genética , Dimetilglicina Desidrogenase , Éxons , Frequência do Gene , Haplótipos , Humanos , Hibridização in Situ Fluorescente , Íntrons , Proteínas Mitocondriais , Dados de Sequência Molecular , Polimorfismo Genético , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: A38-year-old man presented with a history of fish odor (since age 5) and unusual muscle fatigue with increased serum creatine kinase. Our aim was to identify the metabolic error in this new condition. METHODS: We used 1H NMR spectroscopy to study serum and urine from the patient. RESULTS: The concentration of N, N-dimethylglycine (DMG) was increased approximately 100-fold in the serum and approximately 20-fold in the urine. The presence of DMG as a storage product was confirmed by use of 13C NMR spectroscopy and gas chromatography-mass spectrometry. The high concentration of DMG was caused by a deficiency of the enzyme dimethylglycine dehydrogenase (DMGDH). A homozygous missense mutation was found in the DMGDH gene of the patient. CONCLUSIONS: DMGDH deficiency must be added to the differential diagnosis of patients complaining of a fish odor. This deficiency is the first inborn error of metabolism discovered by use of in vitro 1H NMR spectroscopy of body fluids.
Assuntos
Erros Inatos do Metabolismo/enzimologia , Oxirredutases N-Desmetilantes/genética , Adulto , Dimetilglicina Desidrogenase , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Erros Inatos do Metabolismo/sangue , Erros Inatos do Metabolismo/fisiopatologia , Erros Inatos do Metabolismo/urina , Proteínas Mitocondriais , Mutação de Sentido Incorreto , Odorantes , Oxirredutases N-Desmetilantes/deficiência , Oxirredutases N-Desmetilantes/urina , Sarcosina/análogos & derivados , Sarcosina/urinaRESUMO
Dimethylglycine dehydrogenase (DMGDH) (E.C. number 1.5.99.2) is a mitochondrial matrix enzyme involved in the metabolism of choline, converting dimethylglycine to sarcosine. Sarcosine is then transformed to glycine by sarcosine dehydrogenase (E.C. number 1.5.99.1). Both enzymes use flavin adenine dinucleotide and folate in their reaction mechanisms. We have identified a 38-year-old man who has a lifelong condition of fishlike body odor and chronic muscle fatigue, accompanied by elevated levels of the muscle form of creatine kinase in serum. Biochemical analysis of the patient's serum and urine, using (1)H-nuclear magnetic resonance NMR spectroscopy, revealed that his levels of dimethylglycine were much higher than control values. The cDNA and the genomic DNA for human DMGDH (hDMGDH) were then cloned, and a homozygous A-->G substitution (326 A-->G) was identified in both the cDNA and genomic DNA of the patient. This mutation changes a His to an Arg (H109R). Expression analysis of the mutant cDNA indicates that this mutation inactivates the enzyme. We therefore confirm that the patient described here represents the first reported case of a new inborn error of metabolism, DMGDH deficiency.