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.

Recruitment of governing elements for electron transfer in the nitric oxide synthase family.

At least three building blocks are responsible for the molecular basis of the modulation of electron transfer in nitric oxide synthase (NOS) isoforms: the calmodulin-binding sequence, the C-terminal extension, and the autoregulatory loop in the reductase domain. We have attempted to impart the control conferred by the C termini of NOS to cytochrome P450 oxidoreductase (CYPOR), which contains none of these regulatory elements. The effect of these C termini on the properties of CYPOR sheds light on the possible evolutionary origin of NOS and addresses the recruitment of new peptides on the development of new functions for CYPOR. The C termini of NOSs modulate flavoprotein-mediated electron transfer to various electron acceptors. The reduction of the artificial electron acceptors cytochrome c, 2,6-dichlorophenolindophenol, and ferricyanide was inhibited by the addition of any of these C termini to CYPOR, whereas the reduction of molecular O(2) was increased. This suggests a shift in the rate-limiting step, indicating that the NOS C termini interrupt electron flux between flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and/or the electron acceptors. The modulation of CYPOR by the addition of the NOS C termini is also supported by flavin reoxidation and fluorescence-quenching studies and antibody recognition of the C-terminal extension. These experiments support the origin of the NOS enzymes from modules consisting of a heme domain and CYPOR or ferredoxin-NADP(+) reductase- and flavodoxin-like subdomains that constitute CYPOR, followed by further recruitment of smaller modulating elements into the flavin-binding domains.