Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate.

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.

Characterization of osmolyte betaine synthesizing sarcosine dimethylglycine N-methyltransferase from Methanohalophilus portucalensis.

To overcome the extracellular salt stress, Methanohalophilus portucalensis FDF1(T) synthesizes the compatible solute betaine through the methylation of glycine, sarcosine, and N,N-dimethylglycine. S-adenosylmethionine (AdoMet) is the methyl donor. The enzyme sarcosine dimethylglycine N-methyltransferase (SDMT) of M. portucalensis, that catalyzes the formation of N,N-dimethylglycine and glycine betaine, has been purified and characterized. SDMT, a monomer of 33 kDa with a pI at 5.03, has a narrow substrate specificity limited to using only sarcosine and dimethylglycine as substrates for the methyl transferase reaction. The K(m) values for sarcosine and AdoMet were 2.29 and 0.21 mM, respectively, with a V(max) of 0.83 micromol/mg-min (k(cat) value of 0.44 s(-1)). The K(m) values for dimethylglycine and AdoMet were 3.76 and 0.59 mM, respectively, with a V(max) of 4.88 micromol/mg-min (k(cat) of 2.68 s(-1)). A high concentration of the end product betaine (2.0 M) did not affect the SMT activity, but it slightly inhibited the DMT activity. Both activities were also not affected by potassium or sodium ions in concentrations of 200-1,000 mM. We compared this novel archaeal SDMT enzyme to other similar bacterial transferases as well as to the glycine sarcosine dimethylglycine methyltransferase found also in M. portucalensis.

Effects of substrate and potassium on the betaine-synthesizing enzyme glycine sarcosine dimethylglycine N-methyltransferase from a halophilic methanoarchaeon Methanohalophilus portucalensis.

Methanohalophilus portucalensis FDF1 can synthesize the compatible solute betaine de novo through the methylation of glycine, sarcosine and dimethylglycine with the methyl group from S-adenosylmethionine. After separation by DEAE-Sephacel ion chromatography using a KCl step gradient, glycine, sarcosine and dimethylglycine methytransfer (GSDMT) activities were detected in a single peak. The estimated molecular weight of GSDMT was 240 kDa and 2-D gel analysis indicated it was separated into four subunits (52 kDa) with different pI. The PBE94 chromatofocusing column also separated GSDMT into four protein peaks A, B, C, D. Both peak B and D proteins possessed GSDMT activity, while the peak A protein only exhibited SDMT activity. The multiple methyltransferase activities of the large complex appear to be unique compared to other methyltransferases used in betaine synthesis. Further methyltransferase assays in response to different concentrations of KCl indicated that the peak D protein exhibited low GSDMT activity only when K(+) < or = 0.4 M. The peak B protein exhibited a higher GSDMT activity at 0.4 M K(+), while the peak A protein exhibited SDMT activity only at higher K(+) (0.8 M). These results suggest that the internal K(+) concentration regulates GSDMT activities and affects the net betaine accumulation in the cells.