The effect of tetrahydrofolate on the reduction of electron transfer flavoprotein by sarcosine and dimethylglycine dehydrogenases.
ABSTRACT: Pig liver electron transfer flavoprotein (ETF) is rapidly reduced by sarcosine and dimethylglycine dehydrogenases to the anionic semiquinone form, the subsequent formation of the flavoquinol form being a much slower process. In the presence of tetrahydrofolate the yield of anionic semiquinone at the end of the rapid phase of reduction of ETF is only about 10% less than without tetrahydrofolate, as judged by e.p.r. spectroscopy. Tetrahydrofolate does not alter the rate of reduction of ETF by either sarcosine or dimethylglycine dehydrogenase. Nevertheless, it was clearly demonstrated that tetrahydrofolate is a substrate for both sarcosine and dimethylglycine dehydrogenases and is converted to N5,10-methylenetetrahydrofolate.
Project description:An enzyme with sarcosine dimethylglycine methyltransferase (SDMT) activity has been identified in the thermophilic eukaryote, Galdieria sulphuraria. The crystal structure of the enzyme, solved to a resolution of 1.95 A, revealed a fold highly similar to that of mycolic acid synthases. The kcat and apparent K(M) values were 64.3 min(-1) and 2.0 mM for sarcosine and 85.6 min(-1) and 2.8 mM for dimethylglycine, respectively. Apparent K(M) values of S-adenosylmethionine were 144 and 150 microM for sarcosine and dimethylglycine, respectively, and the enzyme melting temperature was 61.1 degrees C. Modeling of cofactor binding in the active site based on the structure of methoxy mycolic acid synthase 2 revealed a number of conserved interactions within the active site.
Project description: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.
Project description: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.
Project description:Sarcosine dimethylglycine methyltransferase (EC 220.127.116.11) is an enzyme from the extremely halophilic anaerobic bacterium Halorhodospira halochoris. This enzyme catalyzes the twofold methylation of sarcosine to betaine, with S-adenosylmethionine (AdoMet) as the methyl-group donor. This study presents the crystallization and preliminary X-ray analysis of recombinant sarcosine dimethylglycine methyltransferase produced in Escherichia coli. Mass spectroscopy was used to determine the purity and homogeneity of the enzyme material. Two different crystal forms, which initially appeared to be hexagonal and tetragonal, were obtained. However, on analyzing the diffraction data it was discovered that both crystal forms were pseudo-merohedrally twinned. The true crystal systems were monoclinic and orthorhombic. The monoclinic crystal diffracted to a maximum of 2.15 A resolution and the orthorhombic crystal diffracted to 1.8 A resolution.
Project description:Sarcosine, a glycine transporter type 1 inhibitor and an N-methyl-D-aspartate (NMDA) receptor co-agonist at the glycine binding site, potentiates NMDA receptor function. Structurally similar to sarcosine, N,N-dimethylglycine (DMG) is also N-methyl glycine-derivative amino acid and commonly used as a dietary supplement. The present study compared the effects of sarcosine and DMG on NMDA receptor-mediated excitatory field potentials (EFPs) in mouse medial prefrontal cortex brain slices using a multi-electrode array system.Glycine, sarcosine and DMG alone did not alter the NMDA receptor-mediated EFPs, but in combination with glutamate, glycine and its N-methyl derivatives significantly increased the frequency and amplitude of EFPs. The enhancing effects of glycine analogs in combination with glutamate on EFPs were remarkably reduced by the glycine binding site antagonist 7-chlorokynurenate (7-CK). However, DMG, but not sarcosine, reduced the frequency and amplitude of EFPs elicited by co-application of glutamate plus glycine. D-cycloserine, a partial agonist at the glycine binding site on NMDA receptors, affected EFPs in a similar manner to DMG. Furthermore, DMG, but not sarcosine, reduced the frequencies and amplitudes of EFPs elicited by glutamate plus D-serine, another endogenous ligand for glycine binding site.These findings suggest that sarcosine acts as a full agonist, yet DMG is a partial agonist at glycine binding site of NMDA receptors. The molecular docking analysis indicated that the interactions of glycine, sarcosine, and DMG to NMDA receptors are highly similar, supporting that the glycine binding site of NMDA receptors is a critical target site for sarcosine and DMG.
Project description:Dimethylglycine dehydrogenase (DMGDH) (E.C. number 18.104.22.168) 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 22.214.171.124). 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.
Project description:Potentiometric titrations of pig liver electron-transfer flavoprotein (ETF) were performed at pH 7.5 and 4 degrees C, both in the reductive and oxidative directions. Reduction of ETF to the hydroquinone form required a total of two reducing equivalents/mol of ETF with the formation of sub-stoichiometric amounts of anionic semiquinone as an intermediate. The oxidation-reduction potentials for the two one-electron couples, oxidized ETF/ETF semiquinone and ETF semiquinone/fully reduced ETF, are +4 mV and -50 mV respectively. The overall midpoint potential for the two-electron couple (oxidized ETF/fully reduced ETF) is -23 mV.
Project description:Here we report crystal structures of dimethylglycine oxidase (DMGO) from the bacterium Arthrobacter globiformis, a bifunctional enzyme that catalyzes the oxidation of N,N-dimethyl glycine and the formation of 5,10-methylene tetrahydrofolate. The N-terminal region binds FAD covalently and oxidizes dimethylglycine to a labile iminium intermediate. The C-terminal region binds tetrahydrofolate, comprises three domains arranged in a ring-like structure and is related to the T-protein of the glycine cleavage system. The complex with folinic acid indicates that this enzyme selectively activates the N10 amino group for initial attack on the substrate. Dead-end reactions with oxidized folate are avoided by the strict stereochemical constraints imposed by the folate-binding funnel. The active sites in DMGO are approximately 40 A apart, connected by a large irregular internal cavity. The tetrahydrofolate-binding funnel serves as a transient entry-exit port, and access to the internal cavity is controlled kinetically by tetrahydrofolate binding. The internal cavity enables sequestration of the reactive iminium intermediate prior to reaction with tetrahydrofolate and avoids formation of toxic formaldehyde. This mode of channelling in DMGO is distinct from other channelling mechanisms.
Project description:Sarcosine (N-methylglycine) is present in many environments inhabited by pseudomonads and is likely most often encountered as an intermediate in the metabolism of choline, carnitine, creatine, and glyphosate. While the enzymology of sarcosine metabolism has been relatively well studied in bacteria, the regulatory mechanisms governing catabolism have remained largely unknown. We previously determined that the sarcosine-catabolic (sox) operon of Pseudomonas aeruginosa is induced by the AraC family regulator GbdR in response to glycine betaine and dimethylglycine. However, induction of these genes was still observed in response to sarcosine in a gbdR deletion mutant, indicating that an independent sarcosine-responsive transcription factor also acted at this locus. Our goal in this study was to identify and characterize this regulator. Using a transposon-based genetic screen, we identified PA4184, or SouR (sarcosine oxidation and utilization regulator), as the sarcosine-responsive regulator of the sox operon, with tight induction specificity for sarcosine. The souR gene is required for appreciable growth on sarcosine as a carbon and nitrogen source. We also characterized the transcriptome response to sarcosine governed by SouR using microarray analyses and performed electrophoretic mobility shift assays to identify promoters directly regulated by the transcription factor. Finally, we characterized PA3630, or GfnR (glutathione-dependent formaldehyde neutralization regulator), as the regulator of the glutathione-dependent formaldehyde detoxification system in P. aeruginosa that is expressed in response to formaldehyde released during the catabolism of sarcosine. This study expands our understanding of sarcosine metabolic regulation in bacteria through the identification and characterization of the first known sarcosine-responsive transcriptional regulator.The Pseudomonas aeruginosa genome encodes many diverse metabolic pathways, yet the specific transcription regulators controlling their expression remain mostly unknown. Here, we used a genetic screen to identify the sarcosine-specific regulator of the sarcosine oxidase operon, which we have named SouR. SouR is the first bacterial regulator shown to respond to sarcosine, and it is required for growth on sarcosine. Sarcosine is found in its free form and is also an intermediate in the catabolic pathways of glycine betaine, carnitine, creatine, and glyphosate. The similarity of SouR to the regulators of carnitine and glycine betaine catabolism suggests evolutionary diversification within this regulatory family to allow response to structurally similar but physiologically distinct ligands.
Project description:Mammalian electron transfer flavoproteins (ETF) are heterodimers containing a single equivalent of flavin adenine dinucleotide (FAD). They function as electron shuttles between primary flavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-A resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the alpha subunit and the third domain is made up entirely by the beta subunit. The N-terminal portion of the alpha subunit and the majority of the beta subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the alpha subunit. Alignment of all the known sequences for the ETF alpha subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of alpha T266, suggesting why the pathogenic mutation, alpha T266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4'-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between alpha H286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins.