Molecular Dissection of the S-Adenosylmethionine-binding Site of Phosphatidylethanolamine N-Methyltransferase

Phosphatidylethanolamine N-methyltransferase (PEMT) is a quatrotopic membrane protein that catalyzes the conversion of phosphatidylethanolamine to phosphatidylcholine through three sequential methylation reactions. Analysis of mice lacking a functional PEMT gene revealed a severe reduction in plasma homocysteine levels. Homocysteine is generated by the hydrolysis of S-adenosylhomocysteine, which is also a product of the PEMT reaction. To gain insight into the PEMT transmethylation reaction and the mechanism by which PEMT regulates homocysteine levels, we sought to define residues that are required for binding of the methyl group donor, S-adenosylmethionine (AdoMet). Bioinformatic analysis of the predicted amino acid sequence of human PEMT identified two putative AdoMet-binding motifs (98GXG100 and 180EE181). Site-directed mutagenesis experiments demonstrated the requirement for the conserved motifs in PEMT specific activity. Analysis of the AdoMet binding ability of mutant recombinant PEMT derivatives established that residues Gly100 and Glu180 are essential for binding of the AdoMet moiety. A significantly elevated KD with respect to AdoMet is observed following conservative mutagenesis of residues Gly98 (400 pmol) and Glu181 (666.7 pmol), relative to the unmodified enzyme (303.1 pmol), suggesting that these residues also participate in AdoMet binding. A model positions two separate AdoMet-binding motifs of PEMT in close proximity at the external leaflet of the endoplasmic reticulum membrane. Phosphatidylethanolamine N-methyltransferase (PEMT) is a quatrotopic membrane protein that catalyzes the conversion of phosphatidylethanolamine to phosphatidylcholine through three sequential methylation reactions. Analysis of mice lacking a functional PEMT gene revealed a severe reduction in plasma homocysteine levels. Homocysteine is generated by the hydrolysis of S-adenosylhomocysteine, which is also a product of the PEMT reaction. To gain insight into the PEMT transmethylation reaction and the mechanism by which PEMT regulates homocysteine levels, we sought to define residues that are required for binding of the methyl group donor, S-adenosylmethionine (AdoMet). Bioinformatic analysis of the predicted amino acid sequence of human PEMT identified two putative AdoMet-binding motifs (98GXG100 and 180EE181). Site-directed mutagenesis experiments demonstrated the requirement for the conserved motifs in PEMT specific activity. Analysis of the AdoMet binding ability of mutant recombinant PEMT derivatives established that residues Gly100 and Glu180 are essential for binding of the AdoMet moiety. A significantly elevated KD with respect to AdoMet is observed following conservative mutagenesis of residues Gly98 (400 pmol) and Glu181 (666.7 pmol), relative to the unmodified enzyme (303.1 pmol), suggesting that these residues also participate in AdoMet binding. A model positions two separate AdoMet-binding motifs of PEMT in close proximity at the external leaflet of the endoplasmic reticulum membrane. PEMT catalyzes the sequential transfer of three methyl groups from AdoMet to phosphatidylethanolamine to generate the essential phospholipid, phosphatidylcholine (PC) 1The abbreviations are: PC, phosphatidylcholine; AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; Hcy, homocysteine; ER, endoplasmic reticulum; PMME, phosphatidylmonomethylethanolamine; PEMT, phosphatidylethanolamine N-methyltransferase; ICMT, isoprenylcysteine carboxyl methyltransferase.1The abbreviations are: PC, phosphatidylcholine; AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; Hcy, homocysteine; ER, endoplasmic reticulum; PMME, phosphatidylmonomethylethanolamine; PEMT, phosphatidylethanolamine N-methyltransferase; ICMT, isoprenylcysteine carboxyl methyltransferase. (1Vance D.E. Ridgway N.D. Prog. Lipid. Res. 1988; 27: 61-79Crossref PubMed Scopus (195) Google Scholar). Concomitant production of one S-adenosylhomocysteine (AdoHcy) molecule occurs with each methylation step (1Vance D.E. Ridgway N.D. Prog. Lipid. Res. 1988; 27: 61-79Crossref PubMed Scopus (195) Google Scholar). The liver is the primary site of PEMT activity, and the PEMT-controlled pathway accounts for ∼30% of hepatic phosphatidylcholine biosynthesis (1Vance D.E. Ridgway N.D. Prog. Lipid. Res. 1988; 27: 61-79Crossref PubMed Scopus (195) Google Scholar, 2DeLong C.J. Shen Y.J. Thomas M.J. Cui Z. J. Biol. Chem. 1999; 274: 29683-29688Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 3Reo N.V. Adinehzadeh M. Toxicol. Appl. Pharmacol. 2000; 164: 113-126Crossref PubMed Scopus (7) Google Scholar, 4Reo N.V. Adinehzadeh M. Foy B.D. Biochim. Biophys. Acta. 2002; 1580: 171-188Crossref PubMed Scopus (90) Google Scholar). The enzymes of the CDP-choline pathway, which are active in all nucleated cells, catalyze the remaining 70% of PC biosynthesis in the liver (2DeLong C.J. Shen Y.J. Thomas M.J. Cui Z. J. Biol. Chem. 1999; 274: 29683-29688Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 3Reo N.V. Adinehzadeh M. Toxicol. Appl. Pharmacol. 2000; 164: 113-126Crossref PubMed Scopus (7) Google Scholar, 4Reo N.V. Adinehzadeh M. Foy B.D. Biochim. Biophys. Acta. 2002; 1580: 171-188Crossref PubMed Scopus (90) Google Scholar, 5Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (190) Google Scholar). In addition to being a key modulator of PC biosynthesis, the liver is the site of ∼85% of all methylation reactions (6Finkelstein J.D. Martin J.J. J. Biol. Chem. 1986; 261: 1582-1587Abstract Full Text PDF PubMed Google Scholar). AdoMet, the primary methyl group donor, is utilized by at least 39 mammalian methyltransferases, including DNA, RNA, protein, lipid, and small molecule methyltransferases (7Fauman E.B. Blumenthal R.M. Cheng X. Cheng X. Blumenthal R.M. S-Adenosylmethionine-Dependent Methyltransferases: Structures and Functions. World Scientific Publishing, Singapore1999: 1-32Crossref Google Scholar, 8Fujioka M. Int. J. Biochem. 1992; 24: 1917-1924Crossref PubMed Scopus (45) Google Scholar). Each AdoMet-dependent transmethylation reaction generates AdoHcy, which in turn is hydrolyzed to yield adenosine and the non-protein amino acid, homocysteine (Hcy) (9Finkelstein J.D. Eur. J. Pediatr. 1998; 157: S40-S44Crossref PubMed Google Scholar). Because mild hyperhomocysteinemia is an independent risk factor for cardiovascular and atherosclerotic disease, circulating plasma homocysteine levels are of significant clinical interest (10Refsum H. Ueland P.M. Curr. Opin. Lipidol. 1998; 9: 533-539Crossref PubMed Scopus (68) Google Scholar). Previous studies on PEMT focused on the PC biosynthetic function of the enzyme and, in particular, whether PEMT-derived PC was targeted to a specific hepatic fate such as very low density lipoprotein particles or bile (11Vance J.E. Vance D.E. J. Biol. Chem. 1986; 261: 4486-4491Abstract Full Text PDF PubMed Google Scholar, 12Nishimaki-Mogami T. Suzuki K. Okochi E. Takahashi A. Biochim. Biophys. Acta. 1996; 1304: 11-20Crossref PubMed Scopus (27) Google Scholar, 13Nishimaki-Mogami T. Suzuki K. Takahashi A. Biochim. Biophys. Acta. 1996; 1304: 21-31Crossref PubMed Scopus (35) Google Scholar, 14Agellon L.B. Walkey C.J. Vance D.E. Kuipers F. Verkade H.J. Hepatology. 1999; 30: 725-729Crossref PubMed Scopus (26) Google Scholar). Recently, however, phenotypic analysis of mice homozygous for a disrupted PEMT allele revealed a novel role for PEMT in the regulation of plasma Hcy levels (15Noga A.A. Stead L.M. Zhao Y. Brosnan M.E. Brosnan J.T. Vance D.E. J. Biol. Chem. 2003; 278: 5952-5955Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Although the liver is the site of numerous AdoMet-dependent methylation reactions, each of which contributes to the Hcy pool, genetic ablation of the PEMT gene alone resulted in a 50% decrease in circulating Hcy levels (15Noga A.A. Stead L.M. Zhao Y. Brosnan M.E. Brosnan J.T. Vance D.E. J. Biol. Chem. 2003; 278: 5952-5955Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Furthermore, hepatoma cells transfected with a cDNA encoding PEMT secreted more Hcy than mock transfected cells (15Noga A.A. Stead L.M. Zhao Y. Brosnan M.E. Brosnan J.T. Vance D.E. J. Biol. Chem. 2003; 278: 5952-5955Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Combined, these results suggest a key yet previously unknown role for PEMT in the regulation of hepatic one-carbon metabolism. To gain further insight into the PEMT transmethylation reaction and the mechanism by which this enzyme modulates plasma Hcy levels, we sought to identify residues that are required to bind AdoMet/AdoHcy. A plethora of enzymes bind AdoMet and/or AdoHcy, including the AdoMet-dependent methyltransferases, AdoMet synthetase, AdoMet decarboxylase, and AdoHcy hydrolase (16Kagan R.M. Clarke S. Arch. Biochem. Biophys. 1994; 310: 417-427Crossref PubMed Scopus (420) Google Scholar). Comparative amino acid sequence analysis previously identified several conserved motifs that bind the AdoMet/AdoHcy moieties, but a small number of methyltransferases including the eukaryotic PEMT family of enzymes do not contain any of these motifs (16Kagan R.M. Clarke S. Arch. Biochem. Biophys. 1994; 310: 417-427Crossref PubMed Scopus (420) Google Scholar, 17Posfai J. Bhagwat A.S. Posfai G. Roberts R.J. Nucleic Acids Res. 1989; 17: 2421-2435Crossref PubMed Scopus (436) Google Scholar, 18Romano J.D. Michaelis S. Mol. Biol. Cell. 2001; 12: 1957-1971Crossref PubMed Scopus (59) Google Scholar). Using bioinformatic analysis, two putative AdoMet/AdoHcy-binding motifs were identified that are conserved among the eukaryotic PEMT proteins. Here, we describe the biochemical evaluation of the motifs in the human PEMT enzyme. Understanding the nature of the interaction between AdoMet/AdoHcy and PEMT will promote resolution of the mechanism by which the enzyme modulates plasma Hcy levels and facilitate the design of agents for therapeutic intervention in cases of hyperhomocysteinemia. Materials—Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, restriction endonucleases, and Platinum Pfx DNA polymerase were from Invitrogen. Oligonucleotides for mutagenesis were synthesized in the DNA core facility at the Department of Biochemistry of the University of Alberta. FuGENE transfection reagent was from Roche Applied Science. S-Adenosyl-l-[methyl-3H]methionine (15 Ci/mmol) was obtained from Amersham Biosciences. S-Adenosyl-l-methionine was from Sigma. HAWP 02500 filters and Multiscreen 96-well filtration/assay plates (MHABN4510) for AdoMet binding assays were from Millipore. Goat anti-rabbit secondary antibodies were purchased from Pierce. All other reagents were of the highest standard commercially available. Bioinformatic Analysis—The PROWL ProteinInfo sequence analysis program (prowl.rockefeller.edu) was used to screen for putative AdoMet-binding motifs in the predicted human PEMT primary structure (accession number, NP_009100). The ALIGN program (based on the ClustalW algorithm) at the San Diego Supercomputer was utilized to the of motifs between eukaryotic PEMT enzymes J.D. Nucleic Acids Res. 1994; PubMed Scopus Google Scholar). PEMT derivatives for analysis of putative AdoMet-binding were generated by the by mutagenesis the as a for all reactions R.M. 1989; PubMed Scopus Google Scholar, C.J. Vance D.E. Biochim. Biophys. Acta. 1999; PubMed Scopus Google Scholar). of the human PEMT to into the and of the mammalian C.J. Vance D.E. Biochim. Biophys. Acta. 1999; PubMed Scopus Google Scholar). is the of a mutant were to and into the and restriction All of the were to of and of the at the of the University of Alberta. encoding conservative were generated as To the at the in the to an and generate the A was with and was with the mutant and and to generate the mutant was with and from A and as Each of the other mutant was by the but a mutant for the of each PEMT To the at the in the to an and generate the was To residues in the to residues and generate the was the mutant of the and mutant the mutant were as and encoding the and were generated as To the at to an and generate the A was with and was with the mutant and and to generate the mutant was with and from A and as To the at to an and generate the a was the mutant was and cells, obtained from the were in modified Eagle's fetal bovine serum, and at with of encoding PEMT and mutant PEMT derivatives was the FuGENE reagent as previously L.B. Vance D.E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). PEMT and assays were as previously as the methyl and as the methyl group N.D. Vance D.E. 1992; PubMed Scopus Google Scholar). of the recombinant PEMT protein and mutant derivatives were as previously an at the L.B. Vance D.E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). assays were on from transfected The were as previously the was in of L.B. Vance D.E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). assays were to a modified of the of Y. C. S. A. 2001; PubMed Scopus Google Scholar). protein was with of (15 Ci/mmol) in for at The binding was a HAWP 02500 on a and was by with of X. The filters were and was by assays were in the of protein to for binding of to the assays on from mock transfected cells AdoMet by other than of from cells mutant PEMT were relative to the binding of from cells the unmodified enzyme. assays were for analysis as protein as was with of AdoMet and the of AdoMet for at in a of was to each and the was to a 96-well and at for plates were on an and each was with of X. filters were of was to each and was by in a binding assays were as for S-adenosylmethionine binding are as the The was utilized to an analysis of to were to the and with the analysis program of a AdoMet-binding catalyzes a of AdoMet-dependent methylation reactions to PC and the AdoHcy AdoMet-binding motifs and are conserved in the of AdoMet-dependent methyltransferases but a small number of methyltransferases including the enzymes of the eukaryotic PEMT family do not contain the three motifs (16Kagan R.M. Clarke S. Arch. Biochem. Biophys. 1994; 310: 417-427Crossref PubMed Scopus (420) Google Scholar). bioinformatic analysis, we identified a sequence that is conserved among the eukaryotic PEMT and Z. Vance J.E. Vance D.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Furthermore, this is any amino is to a DNA AdoMet-binding one of the AdoMet-binding motifs of AdoMet synthetase, as as a in protein and other such as the and J. Bhagwat A.S. Posfai G. Roberts R.J. Nucleic Acids Res. 1989; 17: 2421-2435Crossref PubMed Scopus (436) Google Scholar, M. J. Mol. Biol. 1989; PubMed Scopus Google Scholar, M. A. Biochem. Full Text PDF PubMed Scopus Google Scholar, A. PubMed Scopus Google putative AdoMet binding is conserved in PEMT of conserved three conserved AdoMet-binding motifs in the of acid AdoMet-dependent of the conserved DNA AdoMet-binding of the AdoMet AdoMet-binding of protein and other such as the proteins. The of the in of the human PEMT enzyme the of the conserved amino acid sequence of eukaryotic PEMT The of the in PEMT enzymes from for each protein are of the the role of the conserved residues in binding of the AdoMet each of the was to an by the mutant PEMT and To the of conservative mutagenesis of each on PEMT activity, cells were transfected with encoding PEMT or the mutant PEMT derivatives or mock transfected with and protein was for PEMT activity. of Gly98 to an PEMT by mutagenesis of Gly100 in the recombinant PEMT derivatives with an demonstrated levels of of the recombinant assays of in the of of revealed that conservative mutagenesis of Gly98 results in a elevated with respect to the methyl donor, AdoMet, but not with respect to the methyl and mutagenesis of Gly98 to a of the transmethylation reaction to the AdoMet moiety. The mutant was at all of and AdoMet not of AdoMet whether the in PEMT were to AdoMet binding activity, we transfected cells with the or mutant PEMT or mock transfected with The were for AdoMet binding The was utilized for AdoMet binding assays we previously demonstrated that PEMT is in the L.B. Vance D.E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). from cells the mutant protein a reduction of in AdoMet binding with the unmodified enzyme In conservative mutagenesis of Gly100 the AdoMet binding of the mutant enzyme. To whether the of the mutant recombinant PEMT was a of in AdoMet binding activity, binding assays were with of of Gly98 to an results in a PEMT with an elevated KD with respect to AdoMet, relative to for the unmodified enzyme a specific role for the in binding of the AdoMet is of AdoMet not in AdoMet binding for the Gly100 mutant not suggesting that the of this is essential to the AdoMet binding function of and of the further the role of the residues in the putative AdoMet-binding we mutagenesis of each to or residues to yield the mutant and mutant were generated in which residues were to residues or to and residues To the PEMT of the and cells were transfected with or mutant or mock transfected with The were for PEMT activity. The cells the mutant PEMT derivatives and were as with mock transfected cells In the of this was in to the conservative which a decrease of in PEMT the mutant PEMT derivatives were of PEMT with an the of each PEMT in the of of or AdoMet not in any of the or and of Gly98 AdoMet the of we the of whether the reduction in PEMT of the mutant was to a specific decrease in the AdoMet binding of the recombinant mutant cells were and AdoMet binding assays were as Although the cells the mutant protein not any significant PEMT the mutant protein of AdoMet binding as with the unmodified enzyme assays in the of of AdoMet revealed that the mutant protein an KD with respect to AdoMet, relative to for the unmodified enzyme Combined, these results suggest that the mutant protein is of binding AdoMet, the AdoMet is not for transmethylation the mutant PEMT enzyme is to transfer the methyl group from the AdoMet AdoMet is in such a that the methyl group is not to the transmethylation Furthermore, mutagenesis of Gly98 to the small amino acid, on PEMT and binding and but to the and a significant decrease in binding A and Gly98 to Gly100 or other residues for binding than binding to AdoMet the mutant protein and the mutant PEMT not bind AdoMet, the mutant PEMT protein, in the of of AdoMet not binding not of a AdoMet-binding number of AdoMet-dependent methyltransferases do not contain the AdoMet-binding motifs (16Kagan R.M. Clarke S. Arch. Biochem. Biophys. 1994; 310: 417-427Crossref PubMed Scopus (420) Google Scholar, 17Posfai J. Bhagwat A.S. Posfai G. Roberts R.J. Nucleic Acids Res. 1989; 17: 2421-2435Crossref PubMed Scopus (436) Google Scholar, M. J. Mol. Biol. 1989; PubMed Scopus Google Scholar). Analysis of enzymes that the three AdoMet-binding motifs the PEMT identified a novel J.D. Michaelis S. Mol. Biol. Cell. 2001; 12: 1957-1971Crossref PubMed Scopus (59) Google to in as the of two of A and by a of residues J.D. Michaelis S. Mol. Biol. Cell. 2001; 12: 1957-1971Crossref PubMed Scopus (59) Google Scholar). To whether the is conserved among the eukaryotic PEMT we analysis of the amino acid Although the of A and do not in human PEMT, two specific of the which the are any amino acid Recently, we demonstrated that PEMT is a quatrotopic membrane protein with a that external to the L.B. Vance D.E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). a the to the and the to the external of the membrane. Because AdoMet is in the we that the is more to function in an AdoMet binding in human PEMT S. Biochim. Biophys. Acta. PubMed Scopus (45) Google Scholar). of the PEMT the role of the conserved in binding to AdoMet, we each to an to generate the mutant PEMT and To the conservative we PEMT in from cells that the recombinant PEMT of Glu180 to an PEMT activity, mutagenesis of Glu181 resulted in a 70% decrease in PEMT with an demonstrated levels of of the recombinant of of or AdoMet not to the to levels and assays in the of of AdoMet revealed an elevated with respect to AdoMet in the of the mutant a role for the in an of the transmethylation reaction is of the AdoMet whether the in PEMT to a reduction in AdoMet binding activity, we AdoMet binding assays on transfected of Glu180 to an the AdoMet binding of the recombinant mutant enzyme mutagenesis of Glu181 a protein that a in AdoMet binding assays of the PEMT not binding at any AdoMet not the of Glu180 is essential to the AdoMet binding of of Glu181 results in a PEMT with significantly AdoMet binding ability pmol) relative to the enzyme pmol) Because the in binding and of the mutant are and and the of Glu181 with an a protein with AdoMet binding that the PEMT of the was a of specific in the AdoMet binding activity. a functional role for the in the AdoMet binding of the human PEMT enzyme is AdoMet is the used (7Fauman E.B. Blumenthal R.M. Cheng X. Cheng X. Blumenthal R.M. S-Adenosylmethionine-Dependent Methyltransferases: Structures and Functions. World Scientific Publishing, Singapore1999: 1-32Crossref Google Scholar). The AdoMet is required for the of several essential including PC, and as as for the methylation of DNA and S. K. Homocysteine in and University Scholar). A of the of AdoMet is in that the that the gene encoding AdoMet is that of the S. C. J. Zhao Science. 1998; PubMed Scopus Google Scholar). clinical the AdoMet molecule is with the non-protein amino acid, Hcy, elevated plasma of which are an independent risk factor for cardiovascular (10Refsum H. Ueland P.M. Curr. Opin. Lipidol. 1998; 9: 533-539Crossref PubMed Scopus (68) Google Scholar). To gain insight into the PEMT transmethylation reaction and, the mechanism by which genetic ablation of PEMT circulating plasma homocysteine levels, we the role of specific PEMT amino acid residues in the binding of AdoMet/AdoHcy. identified two motifs that are required for PEMT to bind the methyl AdoMet-binding motifs are conserved in the of the AdoMet-dependent methyltransferases, but these motifs are from several enzymes including of the eukaryotic PEMT family and the isoprenylcysteine carboxyl family of enzymes (16Kagan R.M. Clarke S. Arch. Biochem. Biophys. 1994; 310: 417-427Crossref PubMed Scopus (420) Google Scholar, 18Romano J.D. Michaelis S. Mol. Biol. Cell. 2001; 12: 1957-1971Crossref PubMed Scopus (59) Google Scholar). The enzymes of a in which that in a such as and carboxyl methylation (10Refsum H. Ueland P.M. Curr. Opin. Lipidol. 1998; 9: 533-539Crossref PubMed Scopus (68) Google Scholar). Although from all eukaryotic PEMT the three AdoMet-binding motifs are in the PEMT by the gene (16Kagan R.M. Clarke S. Arch. Biochem. Biophys. 1994; 310: 417-427Crossref PubMed Scopus (420) Google Scholar, C. J. Biol. Chem. Full Text PDF PubMed Google Scholar). this enzyme is a protein that with the PEMT C. J. Biol. Chem. Full Text PDF PubMed Google Scholar, B.D. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). A AdoMet-binding is conserved in the eukaryotic PEMT and mutagenesis of each demonstrated in the AdoMet binding of an Gly100 is essential for binding of the AdoMet Gly98 in a conservative mutagenesis of the was significant binding was Gly98 was with a the KD with respect to AdoMet was elevated in the of each Gly98 a role for this in binding of AdoMet is do not to the that the is also the binding of AdoMet, we demonstrated the role of other PEMT residues in AdoMet binding. an Gly100 is essential for AdoMet binding and the of Gly98 with an a on AdoMet of a with an Gly100 to in AdoMet binding. Because this was not the we a role for Gly98 in the AdoMet binding of the human PEMT enzyme. To further define the AdoMet binding site of human PEMT, we a AdoMet-binding J.D. Michaelis S. Mol. Biol. Cell. 2001; 12: 1957-1971Crossref PubMed Scopus (59) Google Scholar). was following of the family of the and several J.D. Michaelis S. Mol. Biol. Cell. 2001; 12: 1957-1971Crossref PubMed Scopus (59) Google Scholar). in analysis, we that the is also conserved in the and human PEMT analysis of a of the family of methyltransferases, that the enzyme is in the membrane such that the two of the are on the external of the membrane J.D. Michaelis S. Mol. Biol. Cell. 2001; 12: 1957-1971Crossref PubMed Scopus (59) Google Scholar). an the is predicted to the membrane through the of a J.D. Michaelis S. Mol. Biol. Cell. 2001; 12: 1957-1971Crossref PubMed Scopus (59) Google Scholar). The of the enzyme is to a J.D. Michaelis S. Mol. Biol. Cell. 2001; 12: 1957-1971Crossref PubMed Scopus (59) Google Scholar). In the in the human PEMT enzyme is than the amino for and a model on L.B. Vance D.E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). a structure the two of the on of the membrane. in this the of the is and to the L.B. Vance D.E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, S. Biochim. Biophys. Acta. PubMed Scopus (45) Google Scholar). Although we not the that the a role in the AdoMet binding of PEMT, is that the is the functional AdoMet-binding of the in the human PEMT enzyme. of the with AdoMet binding assays the of the with Glu180 in being essential to the AdoMet binding of Glu181 also a role in AdoMet mutagenesis of the results in a significantly elevated KD with respect to such as contain an and a that are by amino PubMed Scopus Google Scholar). between the of such and the group of the adenosine demonstrated PubMed Scopus Google Scholar). or residues in the conserved of PEMT bind the AdoMet through of each to an AdoMet binding activity. Although residues also the of the the AdoMet binding of the residues in this of the human PEMT enzyme. the of the PEMT enzyme and the predicted of the we a model that the conserved and motifs the of the and to the AdoMet in the S. Biochim. Biophys. Acta. PubMed Scopus (45) Google Scholar). the and are the conserved and motifs S-adenosylhomocysteine which AdoHcy to Hcy and is also in the P.M. J. M. PubMed Scopus Google Scholar). the of these the of a mechanism that the AdoMet-dependent PEMT reaction to circulating Hcy levels. In we identified two amino motifs in the human PEMT enzyme that are essential for binding of The AdoMet-binding site of PEMT is in that to the binding motifs but is a novel of Because PEMT to circulating plasma Hcy levels, the enzyme a novel for therapeutic intervention in with hyperhomocysteinemia. of key motifs that bind AdoMet will the design of such therapeutic for are to for

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