title: Mechanisms for auto-inhibition and forced product release in glycine N-methyltransferase: crystal structures of wild-type, mutant R175K and S-adenosylhomocysteine-bound R175K enzymes.
abstract: Glycine N-methyltransferase (S-adenosyl-l-methionine: glycine methyltransferase, EC 2.1.1.20; GNMT) catalyzes the AdoMet-dependent methylation of glycine to form sarcosine (N-methylglycine). Unlike most methyltransferases, GNMT is a tetrameric protein showing a positive cooperativity in AdoMet binding and weak inhibition by S-adenosylhomocysteine (AdoHcy). The first crystal structure of GNMT complexed with AdoMet showed a unique "closed" molecular basket structure, in which the N-terminal section penetrates and corks the entrance of the adjacent subunit. Thus, the apparent entrance or exit of the active site is not recognizable in the subunit structure, suggesting that the enzyme must possess a second, enzymatically active, "open" structural conformation. A new crystalline form of the R175K enzyme has been grown in the presence of an excess of AdoHcy, and its crystal structure has been determined at 3.0 A resolution. In this structure, the N-terminal domain (40 amino acid residues) of each subunit has moved out of the active site of the adjacent subunit, and the entrances of the active sites are now opened widely. An AdoHcy molecule has entered the site occupied in the "closed" structure by Glu15 and Gly16 of the N-terminal domain of the adjacent subunit. An AdoHcy binds to the consensus AdoMet binding site observed in the other methyltransferase. This AdoHcy binding site supports the glycine binding site (Arg175) deduced from a chemical modification study and site-directed mutagenesis (R175K). The crystal structures of WT and R175K enzymes were also determined at 2.5 A resolution. These enzyme structures have a closed molecular basket structure and are isomorphous to the previously determined AdoMet-GNMT structure. By comparing the open structure to the closed structure, mechanisms for auto-inhibition and for the forced release of the product AdoHcy have been revealed in the GNMT structure. The N-terminal section of the adjacent subunit occupies the AdoMet binding site and thus inhibits the methyltransfer reaction, whereas the same N-terminal section forces the departure of the potentially potent inhibitor AdoHcy from the active site and thus facilitates the methyltransfer reaction. Consequently GNMT is less active at a low level of AdoMet concentration, and is only weakly inhibited by AdoHcy. These properties of GNMT are particularly suited for regulation of the cellular AdoMet/AdoHcy ratio.
authors.affiliation: Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045-2106, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Huang
authors.forename: J
authors.initials: J
authors.lastname: Komoto
authors.forename: K
authors.initials: K
authors.lastname: Konishi
authors.forename: Y
authors.initials: Y
authors.lastname: Takata
authors.forename: H
authors.initials: H
authors.lastname: Ogawa
authors.forename: T
authors.initials: T
authors.lastname: Gomi
authors.forename: M
authors.initials: M
authors.lastname: Fujioka
authors.forename: F
authors.initials: F
authors.lastname: Takusagawa
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D016384:Consensus Sequence; D018360:Crystallography, X-Ray; D005998:Glycine / Q000378:metabolism; D050938:Glycine N-Methyltransferase; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D011487:Protein Conformation; D012435:S-Adenosylhomocysteine / Q000737:chemistry / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D008780:Methyltransferases; D050938:Glycine N-Methyltransferase; C494512:Gnmt protein, rat; D005998:Glycine