Structure and biochemical properties of recombinant human dimethylglycine dehydrogenase and comparison to the disease-related H109R variant.

The human dimethylglycine dehydrogenase (hDMGDH) is a flavin adenine dinucleotide (FAD)- and tetrahydrofolate (THF)-dependent, mitochondrial matrix enzyme taking part in choline degradation, one-carbon metabolism and electron transfer to the respiratory chain. The rare natural variant H109R causes dimethylglycine dehydrogenase deficiency leading to increased blood and urinary dimethylglycine concentrations. A detailed biochemical and structural characterization of hDMGDH was thus far hampered by insufficient heterologous expression of the protein. In the present study, we report the development of an intracellular, heterologous expression system in Komagataella phaffii (formerly known as Pichia pastoris) providing the opportunity to determine kinetic parameters, spectroscopic properties, thermostability, and the redox potential of hDMGDH. Moreover, we have successfully crystallized the wild-type enzyme and determined the structure to 3.1-Å resolution. The structure-based analysis of our biochemical data provided new insights into the kinetic properties of the enzyme in particular with respect to oxygen reactivity. A comparative study with the H109R variant demonstrated that the variant suffers from decreased protein stability, cofactor saturation, and substrate affinity. DATABASE: Structural data are available in the PDB database under the accession number 5L46.

Folate in demethylation: the crystal structure of the rat dimethylglycine dehydrogenase complexed with tetrahydrofolate.

Dimethylglycine dehydrogenase (DMGDH) is a mammalian mitochondrial enzyme which plays an important role in the utilization of methyl groups derived from choline. DMGDH is a flavin containing enzyme which catalyzes the oxidative demethylation of dimethylglycine in vitro with the formation of sarcosine (N-methylglycine), hydrogen peroxide and formaldehyde. DMGDH binds tetrahydrofolate (THF) in vivo, which serves as an acceptor of formaldehyde and in the cell the product of the reaction is 5,10-methylenetetrahydrofolate instead of formaldehyde. To gain insight into the mechanism of the reaction we solved the crystal structures of the recombinant mature and precursor forms of rat DMGDH and DMGDH-THF complexes. Both forms of DMGDH reveal similar kinetic parameters and have the same tertiary structure fold with two domains formed by N- and C-terminal halves of the protein. The active center is located in the N-terminal domain while the THF binding site is located in the C-terminal domain about 40Å from the isoalloxazine ring of FAD. The folate binding site is connected with the enzyme active center via an intramolecular channel. This suggests the possible transfer of the intermediate imine of dimethylglycine from the active center to the bound THF where they could react producing a 5,10-methylenetetrahydrofolate. Based on the homology of the rat and human DMGDH the structural basis for the mechanism of inactivation of the human DMGDH by naturally occurring His109Arg mutation is proposed.

Molecular basis of dimethylglycine dehydrogenase deficiency associated with pathogenic variant H109R.

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 micromol/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.

The purified recombinant precursor of rat mitochondrial dimethylglycine dehydrogenase binds FAD via an autocatalytic reaction.

The precursor of the rat mitochondrial flavoenzyme dimethylglycine dehydrogenase (Me(2)GlyDH) has been produced in Escherichia coli as a C-terminally 6-His-tagged fusion protein, purified by one-step affinity chromatography and identified by ESI-MS/MS. It was correctly processed into its mature form upon incubation with solubilized rat liver mitoplasts. The purified precursor was mainly in its apo-form as demonstrated by immunological and fluorimetric detection of covalently bound flavin adenine dinucleotide (FAD). Results described here definitively demonstrate that: (i) covalent attachment of FAD to Me(2)GlyDH apoenzyme can proceed in vitro autocatalytically, without third reactants; (ii) the removal of mitochondrial presequence by mitochondrial processing peptidase is not required for covalent autoflavinylation.