ABSTRACT
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.
Subject(s)
Metabolism, Inborn Errors/enzymology , Metabolism, Inborn Errors/genetics , Oxidoreductases, N-Demethylating/deficiency , Oxidoreductases, N-Demethylating/genetics , Point Mutation/genetics , Sarcosine/analogs & derivatives , Adult , Amino Acid Sequence , Amino Acid Substitution/genetics , Base Sequence , Black People/genetics , Blotting, Western , Cell Line , Chronic Disease , Cloning, Molecular , Creatine Kinase/blood , DNA Mutational Analysis , Dimethylglycine Dehydrogenase , Expressed Sequence Tags , Fatigue/complications , Fatigue/enzymology , Fatigue/genetics , Fatigue/metabolism , Humans , Magnetic Resonance Spectroscopy , Male , Metabolism, Inborn Errors/complications , Metabolism, Inborn Errors/metabolism , Mitochondria/enzymology , Mitochondrial Proteins , Molecular Sequence Data , Odorants , Oxidoreductases, N-Demethylating/chemistry , Phenotype , Sarcosine/blood , Sarcosine/urineABSTRACT
Cofactor associations within the electron transferring flavoprotein (ETF) were studied in real time using microelectrospray ionization-mass spectrometry (muESI-MS). Initial analysis of porcine (pETF) and human ETF (hETF) revealed only the holoprotein. When muESI-MS source energies were increased, both pETF and hETF readily lost AMP. Analysis of hETF and pETF in methanol revealed intact alpha- and beta-subunits, and beta-subunit with AMP. The pETF also contained beta-subunit with FAD and beta-subunit with both cofactors. In contrast to crystal structure predictions, AMP dissociates more readily than FAD, and the pETF beta-subunit has an intimate association with FAD. This work demonstrates the complementarity of muESI-MS with NMR X-ray and optical spectroscopy in the analysis of noncovalent complexes.