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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.
Copyright © 2014 Elsevier Inc. All rights reserved.
An important epigenetic modification is the methylation/demethylation of histone lysine residues. The first histone demethylase to be discovered was a lysine-specific demethylase 1, LSD1, a flavin containing enzyme which carries out the demethylation of di- and monomethyllysine 4 in histone H3. The removed methyl groups are oxidized to formaldehyde. This reaction is similar to those performed by dimethylglycine dehydrogenase and sarcosine dehydrogenase, in which protein-bound tetrahydrofolate (THF) was proposed to serve as an acceptor of the generated formaldehyde. We showed earlier that LSD1 binds THF with high affinity which suggests its possible participation in the histone demethylation reaction. In the cell, LSD1 interacts with co-repressor for repressor element 1 silencing transcription factor (CoREST). In order to elucidate the role of folate in the demethylating reaction we solved the crystal structure of the LSD1-CoREST-THF complex. In the complex, the folate-binding site is located in the active center in close proximity to flavin adenine dinucleotide. This position of the folate suggests that the bound THF accepts the formaldehyde generated in the course of histone demethylation to form 5,10-methylene-THF. We also show the formation of 5,10-methylene-THF during the course of the enzymatic reaction in the presence of THF by mass spectrometry. Production of this form of folate could act to prevent accumulation of potentially toxic formaldehyde in the cell. These studies suggest that folate may play a role in the epigenetic control of gene expression in addition to its traditional role in the transfer of one-carbon units in metabolism.
© 2014 The Protein Society.
Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. In mammalian liver it reduces S-adenosylmethionine levels by using it to methylate glycine, producing N-methylglycine (sarcosine) and S-adenosylhomocysteine. GNMT is inhibited by binding two molecules of 5-methyltetrahydrofolate (mono- or polyglutamate forms) per tetramer of the active enzyme. Inhibition is sensitive to the status of the N-terminal valine of GNMT and to polyglutamation of the folate inhibitor. It is inhibited by pentaglutamate form more efficiently compared to monoglutamate form. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein expressed in E. coli where the N-terminus is not acetylated. In this work we used a protein crystallography approach to evaluate the structural basis for these differences. We show that in the folate-GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate-recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme.
Copyright © 2011 Elsevier B.V. All rights reserved.
Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. It is abundant in the liver, where it uses excess S-adenosylmethionine (AdoMet) to methylate glycine to N-methylglycine (sarcosine) and produces S-adenosylhomocysteine (AdoHcy), thereby controlling the methylating potential of the cell. GNMT also links utilization of preformed methyl groups, in the form of methionine, to their de novo synthesis, because it is inhibited by a specific form of folate, 5-methyltetrahydrofolate. Although the structure of the enzyme has been elucidated by x-ray crystallography of the apoenzyme and in the presence of the substrate, the location of the folate inhibitor in the tetrameric structure has not been identified. We report here for the first time the crystal structure of rat GNMT complexed with 5-methyltetrahydrofolate. In the GNMT-folate complex, two folate binding sites were located in the intersubunit areas of the tetramer. Each folate binding site is formed primarily by two 1-7 N-terminal regions of one pair of subunits and two 205-218 regions of the other pair of subunits. Both the pteridine and p-aminobenzoyl rings are located in the hydrophobic cavities formed by Tyr5, Leu207, and Met215 residues of all subunits. Binding experiments in solution also confirm that one GNMT tetramer binds two folate molecules. For the enzymatic reaction to take place, the N-terminal fragments of GNMT must have a significant degree of conformational freedom to provide access to the active sites. The presence of the folate in this position provides a mechanism for its inhibition.
Glycine N-methyltransferase (EC 22.214.171.124) catalyzes the methylation of glycine by S-adenosylmethionine to form sarcosine and S-adenosylhomocysteine. The enzyme was previously shown to be abundant in both the liver and pancreas of the rat, to consist of four identical monomers, and to contain tightly bound folate polyglutamates in vivo. We now report that the inhibition of glycine N-methyltransferase by (6S)-5-CH(3)-H(4)PteGlu(5) is noncompetitive with regard to both S-adenosylmethionine and glycine. The enzyme exhibits strong positive cooperativity with respect to S-adenosylmethionine. Cooperativity increases with increasing concentrations of 5-CH(3)-H(4)PteGlu(5) and is greater at physiological pH than at pH 9.0, the pH optimum. Under the same conditions, cooperativity is much greater for the pancreatic form of the enzyme. The V(max) for the liver form of the enzyme is approximately twice that of the pancreatic enzyme, while K(m) values for each substrate are similar in the liver and pancreatic enzymes. For the liver enzyme, at pH 7.0 half-maximal inhibition is seen at a concentration of about 0.2 microM (6S)-5-CH(3)-H(4)PteGlu(5), while at pH 9.0 this value is increased to about 1 microM. For the liver form of the enzyme, 50% inhibition with respect to S-adenosylmethionine at pH 7.4 occurs at about 0.27 microM. The dissociation constant, K(s), obtained from binding data at pH 7.4 is 0.095. About 1 mol of (6S)-5-CH(3)-H(4)PteGlu(5) was bound per tetramer at pH 7.0, and 1.6 mol were bound at pH 9.0. The degree of binding and inhibition were closely parallel at each pH. At equal concentrations of (6R,6S)- and (6S)-5-CH(3)-H(4)PteGlu(5), the natural (6S) form was about twice as inhibitory. These studies indicate that glycine N-methyltransferase is a highly allosteric enzyme, which is consistent with its role as a regulator of methyl group metabolism in both the liver and the pancreas.
Glycine N-methyltransferase (GNMT) is inhibited by 5-methyltetrahydrofolate polyglutamate in vitro. It is believed to play a regulatory role in the synthesis de novo of methyl groups. We have used the amino-acid-defined diet of Walzem and Clifford [(1988) J. Nutr. 118, 1089-1096] to determine whether folate deficiency in vivo would affect GNMT activity, as predicted by the studies in vitro. Weanling male rats were fed on the folate-deficient diet or a folate-supplemented diet pair-fed to the deficient group. A third group was fed on the folate-supplemented diet ad libitum. Development of folate deficiency rapidly resulted in decreased levels of S-adenosylmethionine (SAM) and elevation of S-adenosylhomocysteine (SAH). The ratios of SAM to SAH were 1.8, 2.7 and 1.5 in the deficient group for weeks 2, 3 and 4 of the experiment, and the values were 9.7, 7.1 and 8.9 for the pair-fed control group and 10.3, 8.8 and 8.0 for the control group ad libitum fed. The activity of GNMT was significantly higher in the deficient group than in either of the two control groups at each time period. This was not due to increased amounts of GNMT protein, but reflected an increase in specific enzyme activity. Levels of folate in both the cytosol and mitochondria were severely lowered after only 2 weeks on the diet. The distribution of folate coenzymes was also affected by the deficiency, which resulted in a marked increase in the percentage of tetrahydrofolate polyglutamates in both cytosol and mitochondria and a very large decrease in cytosolic 5-methyltetrahydrofolate. The increased GNMT activity is therefore consistent with decreased folate levels and decreased inhibition of enzyme activity.
An amino acid-defined, folate-deficient diet was used to investigate the regulation of pancreatic glycine N-methyltransferase in vivo. This enzyme modulates the ratio of S-adenosylmethionine to S-adenosylhomocysteine and is inhibited by bound folate in vitro. Rats were fed either a folate-deficient diet, a folate-supplemented diet (pair-fed to the deficient group), or a folate supplemented diet ad libitum and measurements were made after 2, 3, and 4 wk. Folate concentrations were greatly reduced in the folate-deficient pancreas after only 2 wk and pancreatic glycine N-methyltransferase activity was elevated but the amount of immunologically measured enzyme protein was the same. The ratio of S-adenosylmethionine to S-adenosylhomocysteine was rapidly reduced in the deficient pancreas. This ratio was also reduced with age in the ad libitum control rats. The pancreas of deficient rats had more immature secretory granules and the ducts were devoid of secreted material.
10-Formyltetrahydrofolate dehydrogenase (EC 126.96.36.199) was previously identified as a folate-binding protein in rat liver cytosol (R.J. Cook and C. Wagner, Biochemistry 21, 4427-4434, 1982) by virtue of the tetrahydrofolate polyglutamate tightly bound to the partially purified enzyme. In this current study we provide evidence to show that when liver cytosol was rapidly processed to identify the protein bound folate, large amounts of both 10-formyl- and 5-formyltetrahydrofolate were present. After overnight storage of the cytosol at 5 degrees C before processing, almost no formylfolates were present and the major protein-bound form was tetrahydrofolate. This suggests that 10-formyltetrahydrofolate polyglutamates are tightly bound to the enzyme in vivo and are converted to tetrahydrofolate forms during isolation by the hydrolase activity associated with the enzyme. Covalent binding of the stable folate analogue, 5-formyltetrahydrofolate, to the purified enzyme resulted in 2 mol bound per mole of enzyme subunit. This is consistent with earlier reports suggesting the enzyme is capable of carrying out both oxidative and hydrolytic conversion of 10-formyltetrahydrofolate to tetrahydrofolate at the same time. Partial tryptic digestion of the purified enzyme selectively inhibited dehydrogenase activity of the enzyme but did not affect the hydrolase or aldehyde dehydrogenase activities.
The high molecular weight folate binding protein of rat liver cytosol has been purified to apparent homogeneity. Purification was achieved by using a combination of gel filtration, O-(diethylaminoethyl)cellulose chromatography, and affinity chromatography. This folate binding protein was initially identified during purification by an in vivo labeling procedure involving intraperitoneal injection of [3H]folic acid prior to sacrifice and subsequently by its ability to bind naturally reduced [3H]folate polyglutamates in vitro. A molecular weight of 210 000 was estimated by gel chromatography. This is distinct from the trifunctional formyl-methenyl-methylene synthetase of rat liver which has a molecular weight of 225 000. Sodium dodecyl sulfate electrophoresis revealed a single band with a molecular weight of about 100 000 which suggests the native protein is composed of two identical subunits. The partially purified protein contains bound tetrahydropteroylpentaglutamate.