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.
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. z.luka@vanderbilt.edu
Zigmund
Z
Luka
Svetlana
S
Pakhomova
Lioudmila V
LV
Loukachevitch
Marcia E
ME
Newcomer
Conrad
C
Wagner
D000818:Animals; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D050938:Glycine N-Methyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006860:Hydrogen Bonding; D008099:Liver / Q000201:enzymology*; D008958:Models, Molecular; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D013763:Tetrahydrofolates / Q000737:chemistry / Q000378:metabolism; D014633:Valine / Q000378:metabolism
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013763:Tetrahydrofolates; D050938:Glycine N-Methyltransferase; C494512:Gnmt protein, rat; D014633:Valine; C005984:5-methyltetrahydrofolate