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Molecular basis of dimethylglycine dehydrogenase deficiency associated with pathogenic variant H109R
Author(s) -
McAndrew R. P.,
Vockley J.,
Kim J.J. P.
Publication year - 2008
Publication title -
journal of inherited metabolic disease
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.462
H-Index - 102
eISSN - 1573-2665
pISSN - 0141-8955
DOI - 10.1007/s10545-008-0999-2
Subject(s) - flavoprotein , flavin adenine dinucleotide , biochemistry , cofactor , pyridoxamine , enzyme , oxidoreductase , sarcosine , chemistry , flavin group , mitochondrial matrix , dehydrogenase , biology , microbiology and biotechnology , amino acid , cytosol , glycine , pyridoxal
Summary Dimethylglycine dehydrogenase (DMGDH) is a mitochondrial matrix flavoprotein that catalyses the demethylation of dimethylglycine to form sarcosine, accompanied by the reduction of the covalently bound FAD cofactor. Electron‐transfer flavoprotein reoxidizes the reduced flavin and transfers reducing equivalents to the main mitochondrial respiratory chain through the enzyme ETF‐ubiquinone oxidoreductase. DMGDH plays a prominent role in choline and 1‐carbon metabolism. We have expressed the mature form of human DMGDH and the H109R variant identified in a DMGDH‐deficient patient as N‐terminally His 6 ‐tagged proteins in E. coli . The enzymes were purified to homogeneity by nickel affinity and anion exchange chromatography. The presence of FAD in the wild‐type enzyme was confirmed by spectrophotometric analysis. The H109R variant, however, had only 47% of the wild‐type level of bound flavin as expressed in E. coli , indicating its reduced affinity for FAD As previously described for rat enzyme studies, the wild‐type human enzyme exhibited two K m values for N,N ‐dimethylglycine ( K m1 = 0.039 ± 0.010 mmol/L and K m2 = 15.4 ± 1.2 mmol/L). The addition of 4 μmol/L tetrahydrofolate resulted in a slight decrease in specific activity and a substantial decrease in K m2 (1.10 ± 0.55 mmol/L). The flavinated H109R variant protein exhibited a 27‐fold decrease in specific activity and a 65‐fold increase in K m , explaining its pathogenicity. Additionally, the current expression system represents a significant improvement over a previously described rat DMGDH expression system and will enhance our ability to further study this important metabolic enzyme.