Glycine-N-methyltransferase (GNMT)

Genetics and enzyme structure

Ryan Hughes

200802692

Glycine N-methyltransferase catalyzes the synthesis of sarcosine from glycine using S-

adenosylmethionine as the methyl donor. The action of Gycine-N-methyltransferase (GNMT)

was first described in the literature in 1960. It was proposed that the enzyme, in guinea pig liver,

was involved in “oxidation of methyl carbon of methionine” (1) via oxidative cleavage of sarco-

sine to glycine. However, not all methionine is metabolized in this way, indicating that the en-

zyme may be moonlighting in a role other than methionine catabolism. Later it was shown that

GNMT was critical to the regulation of the relative levels of S-adenosylmethionine (SAM) and S-

adenosylhomocystein (SAH) (2). GNMT was then shown to be a homo-tetramer with a relative

molecular weight of ~130,000. This property was found using equilibrium sedimentation velocity,

chromotography, denaturants, proteases, and Edman degradation. This was also the first time

that GNMT, from rat liver, was isolated (3). The nucleotide sequence of a cloned cDNA was

used to determine that each subunit contains 292 amino acid (AA) residues (4).

An antiserum prepared against a known folate binding protein greatly decreased

GNMT’s activity, implementing it as a folate binding protein (4). The structure of GNMT (with

bound substrates) was found, using X-ray crystallography, for the first time in1996 (5). The crys-

tal structure has similarities to other folate binding protein structures. However, the substrates,

or alosteric activators/inhibitors, may be more diverse. This is due to a molecular basket struc-

ture formed by each GNMT subunit which is capped by the N-terminal AA of an adjacent sub-

unit (6). Apo-GNMT structure was also solved and differs from the GNMT, in the bound form, by

changes in the binding pocket (6). GNMT was found to be inhibited by 5-Methyltetrahydrofolate

Pentaglutamate. This indicates “indirect” product inhibition (negative feedback) because the

level of 5-Methyltetrahydrofolate Pentaglutamate indicates the overall methylating potential of

the cell (7).

The human GNMT gene was isolated from a taiwanese liver cDNA library (8). Subsequently,

this cDNA was radiolabeled and used to probe a human placental genomic DNA library con-

structed in lambda phage FIX II (8). Clones were isolated and sub-cloned into pBluscript II KS.

Sub clones were sequenced, primers were made, and primer extension was used to determine

the transcription start site (TSS) (8). The gene was then analyzed using NCBI Blast and similar

programs. The gene was found to span ~10 kb and consist of six exons (218, 128, 117, 143,

122, and 346 bp) (8). Four Sp1 binding sites and a putative CAAT box were located upstream of

the promoter region (8). Somatic cell hybrids and fluorescent in situ hybridization (FISH) was

used to localize the gene to the short arm of chromosome 6, near the centromere, (6p12).

Northern blotting showed that GNMT is only expressed in liver, pancreas, and prostate (8).

A novel inborn error of metabolism has been described whereby two Italian siblings were

found to be compound heterozygotes for mutations in the GNMT gene. An accumulation of me-

thionine (hypermethionemia) and SAM and a decrease in SAH implicated GNMT deficiency as

the culprit (9). Since the location and sequence of the gene was known, primers could be made

which anneal to the flanking regions of the gene (or exons). The mutated gene from the ge-

nomic DNA of the affected siblings and parents (and unmutated controls) were amplified by

PCR and sequenced (10). It was found that exon 1 from the mother had a transition mutation at

nt 1481, causing the change (Leu49Pro) in the gene product (enzyme). Exon 4 from the father

had a transversion at nt 3715 causing the change (His176Asn). Both the mother and father

were heterozygous for their respective mutations and showed no symptoms of hypermethione-

mia. Their progeny were found to be compound heterozygous for both mutations (10). The en-

zyme functions as a homo-tetramere. Therefore, one who is heterozygous for a recessive, loss

of function, mutation still has one allele which codes for a functional enzyme subunit. However,

one who is compound heterozygous for two different loss of function mutations has four non-

functional subunits. Hence, the progeny are genetically null with respect to the GNMT gene.

The change from leucine to proline in the middle of an alpha helix will cause a major change in

the conformation and stability of the helix, and will most likely affect the function of the protein

(10). Proline is a secondary amine. Upon forming a peptide bond, it has no hydrogen left to do-

nate towards hydrogen bonding. The lack of H-bonding, and lack of flexibility destabilizes the

helix. Likewise, the change from histidine to asparagine will cause pronounced changes in the

secondary structure. The histidine occurs in a beta strand one residue from an arginine which is

implicated in glycine binding (10). Histidine could act as a nucleophile or may be important for

flexibility at the active site (10). Both of the amino acid changes found in the compound het-

erozygous siblings cause the subunits to be inactive, leaving the homo-tetramer enzymatically

inactive. The fact that GNMT is the only known tetrameric SAM dependant methyl transferase

may indicate its role as a regulatory enzyme (10).

A five step mechanism by which GNMT catalyzes the methyl transfer, from SAM to

glycine, was proposed (11). (A) Initial stage: residues 1-8 are in solution. Residues 9-20 form a

U- loop which enters the SAM binding site, of an adjacent subunit, leading to a closed confor-

mation at low SAM concentration. KmSAM is higher than for other methyl transferases due to com-

petition between the U- loop and SAM. This leads to a higher SAM/SAH ratio than would occur

if the enzyme was a single subunit. This ultimately affects the activity of other methyl trans-

ferases (increased substrate concentration). (B) SAM Binding Stage: At higher concentrations of

SAM the U-loop is displaced by SAM (equilibrium is shifted by law of mass action) and the open

complex is formed. In the open complex, hydrogen bonding and pi-pi interactions position SAM.

(C) Gly Binding Stage: The carboxylate group of glycine forms two hydrogen binds to the guani-

dino group of Arg175. The Arg side chain is flexible but five additional hydrogen bonds hold

glycine to its cognate binding pocket. The amino group of glycine is now in close proximity to the

methyl group of SAM. (D) Near the Transition State: The methyl group of SAM is lined up with

the lone pare of electrons from the glycine amino group. The two species are held in place with

hydrogen bonds and the positive charge on the sulphur atom is stabilized. At this point thermal

motion leads to the SN2 reaction whereby the lone pare attacks the methyl group. The electrons,

bonding the methyl group to the sulphur atom, become localized to the sulphur atom. The

methyl transfer has taken place. (E) Product Releasing Stage: Glycine (sarcosine) now has a

methyl group which destabilizes hydrogen bonding to the nitrogen. The positive charge on the

nitrogen destabilizes the charge-charge interaction between the guanidino group go Arg175 and

the carboxyl group of the newly formed sarcosine. The sulphur atom of SAM is now neutral and

no longer interacts, in charge-dipole manner, with the hydroxyl of Tyr21. (F) Final Stage: The

conformational change, brought about by the loss of non-covalent bonding between GNMT and

SAM, brings the U-loop back near the substrate binding site. SAH is not as tightly bound to the

enzyme as SAM, due to loss many non-covalent interactions, and the U-loop may displace the

bound product (11).

The study of GNMT is nowhere near exhausted. The 5’ regulatory region of the GNMT

gene has been more fully characterized recently (12). The TSS was found to be located “at the

14th position upstream of the ATG codon”. NF-Y binding site, SP1 binding site, and an inducible

XRE was identified in the regulatory region. Potential androgen response elements have also

been identified (13). EMSA and ChIP were used to prove that one androgen receptor (AR) in-

teracts with the first exon of the GNMT gene in vitro and in vivo. GNMT has even been impli-

cated in neurogenesis and cognitive performance (14). A decrease in GNMT was found to lead

to cognitive impairment in mice. GNMT and other enzymes of folate mediated one carbon me-

tabolism may also make their way into the nucleus of cells in response to folate deficiency (15).

Thus, even enzymes lacking NLS may somehow become concentrated in the nucleus in a last-

ditch effort to protect the fidelity of DNA synthesis and allow proliferation of the cell line.

References

1. Blumenstein, J. (1960) . Biochemical and Biophysical Research Communi-cations. 3, 259-263

2. Heady, J.E., and Kerr, S.J. (1975) Alteration of glycine N-methyltransferase activity in fetal, adult, and tumor tissues. Cancer Res. 35, 640-643

3. Ogawa, H., and Fujioka, M. (1982) Purification and properties of glycine N-methyltransferase from rat liver. J.Biol.Chem. 257, 3447-3452

4. Cook, R.J., and Wagner, C. (1984) Glycine N-methyltransferase is a folate binding protein of rat liver cytosol. Proc.Natl.Acad.Sci.U.S.A. 81, 3631-3634

5. Fu, Z., Hu, Y., Konishi, K., Takata, Y., Ogawa, H., Gomi, T., Fujioka, M., and Takusagawa, F. (1996) Crystal structure of glycine N-methyltransferase from rat liver. Biochemistry. 35, 11985-11993

6. Pattanayek, R., Newcomer, M.E., and Wagner, C. (1998) Crystal structure of apo-glycine N-methyltransferase (GNMT). Protein Sci. 7, 1326-1331

7. Yeo, E.J., Briggs, W.T., and Wagner, C. (1999) Inhibition of glycine N-methyltransferase by 5-methyltetrahydrofolate pentaglutamate. J.Biol.Chem. 274, 37559-37564

8. Chen, Y.M., Chen, L.Y., Wong, F.H., Lee, C.M., Chang, T.J., and Yang-Feng, T.L. (2000) Genomic structure, expression, and chromosomal localization of the human glycine N-methyltransferase gene. Genomics. 66, 43-47

9. Mudd, S.H., Cerone, R., Schiaffino, M.C., Fantasia, A.R., Minniti, G., Caruso, U., Lorini, R., Watkins, D., Matiaszuk, N., Rosenblatt, D.S., Schwahn, B., Rozen, R., LeGros, L., Kotb, M., Capdevila, A., Luka, Z., Finkelstein, J.D., Tangerman, A., Stabler, S.P., Allen, R.H., and Wagner, C. (2001) Glycine N-methyltransferase deficiency: a novel inborn error causing persistent isolated hypermethioninaemia. J.Inherit.Metab.Dis. 24, 448-46410. Luka, Z., Cerone, R., Phillips, J.A.,3rd, Mudd, H.S., and Wagner, C. (2002) Mutations in human glycine N-methyltransferase give insights into its role in methionine metabolism. Hum.Genet. 110, 68-74

11. Takata, Y., Huang, Y., Komoto, J., Yamada, T., Konishi, K., Ogawa, H., Gomi, T., Fujioka, M., and Takusagawa, F. (2003) Catalytic mechanism of glycine N-methyltransferase. Biochemistry. 42, 8394-8402

12. Lee, C.M., Shih, Y.P., Wu, C.H., and Chen, Y.M. (2009) Characterization of the 5' regulatory region of the human Glycine N-methyltransferase gene. Gene. 443, 151-157

13. Lee, C.M., Yen, C.H., Tzeng, T.Y., Huang, Y.Z., Chou, K.H., Chang, T.J., and Arthur Chen, Y.M. (2013) Androgen response element of the glycine N-methyltransferase gene is located in the coding region of its first exon. Biosci.Rep. 33, 10.1042/BSR20130030

14. Carrasco, M., Rabaneda, L.G., Murillo-Carretero, M., Ortega-Martinez, S., Martinez-Chantar, M.L., Woodhoo, A., Luka, Z., Wagner, C., Lu, S.C., Mato,

J.M., Mico, J.A., and Castro, C. (2014) Glycine N-methyltransferase expression in the hippocampus and its role in neurogenesis and cognitive performance. Hippocampus. 24, 840-852

15. Field, M.S., Kamynina, E., Agunloye, O.C., Liebenthal, R.P., Lamarre, S.G., Brosnan, M.E., Brosnan, J.T., and Stover, P.J. (2014) Nuclear enrichment of fo-late cofactors and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) protect de novo thymidylate biosynthesis during folate deficiency. J.Bi-ol.Chem.