Crystal Structure of Glycine N-Methyltransferase from Rat Liver ²,‡ Zhuji Fu, § Yongbo Hu, § Kiyoshi Konishi, | Yoshimi Takata, | Hirofumi Ogawa, | Tomoharu Gomi, | Motoji Fujioka,* ,| and Fusao Takusagawa* ,§ Department of Biochemistry, UniVersity of Kansas, Lawrence, Kansas 66045, and Department of Biochemistry, Faculty of Medicine, Toyama Medical and Pharmaceutical UniVersity, Sugitani, Toyama 930-01, Japan ReceiVed May 3, 1996; ReVised Manuscript ReceiVed July 3, 1996 X ABSTRACT: Glycine N-methyltransferase (GNMT) from rat liver is a tetrameric enzyme with 292 amino acid residues in each identical subunit and catalyzes the S-adenosylmethionine (AdoMet) dependent methylation of glycine to form sarcosine. The crystal structure of GNMT complexed with AdoMet and acetate, a competitive inhibitor of glycine, has been determined at 2.2 Å resolution. The subunit of GNMT forms a spherical shape with an extended N-terminal region which corks the entrance of active site of the adjacent subunit. The active site is located in the near center of the spherical subunit. As a result, the AdoMet and acetate in the active site are completely surrounded by amino acid residues. Careful examination of the structure reveals several characteristics of GNMT. (1) Although the structure of the AdoMet binding domain of the GNMT is very similar to those of other methyltransferases recently determined by X-ray diffraction method, an additional domain found only in GNMT encloses the active site to form a molecular basket, and consequently the structure of GNMT looks quite different from those of other methyltransferases. (2) This unique molecular structure can explain why GNMT can capture folate and polycyclic aromatic hydrocarbons. (3) The unique N-terminal conformation and the subunit structure can explain why GNMT exhibits positive cooperativity in binding AdoMet. From the structural features of GNMT, we propose that the enzyme might be able to capture yet unidentified molecules in the cytosol and thus participates in various biological processes including detoxification of polycyclic aromatic hydrocarbons. In the active site, acetate binds near the S-CH 3 moiety of AdoMet. Simple modeling indicates that the amino group of the substrate glycine can be placed close to the methyl group of AdoMet within 3.0 Å and form a hydrogen bond with the carboxyl group of Glu 15 of the adjacent subunit. On the basis of the ternary complex structure, the mechanism of the methyl transfer in GNMT has been proposed. Biological methylation reactions utilizing S-adenosylme- thionine (AdoMet) as the methyl donor are widespread in nature and participate in a wide variety of cellular processes through methylation of nucleic acids, proteins, phospholipids, and small molecules. Small molecule methyltransferases may be divided into two categories: (1) the enzymes involved in the biosynthesis and degradation of bioactive amines, e.g., norepinephrine N-methyltransferase, acetylse- rotonin methyltransferase, and catechol O-methyltransferase, and (2) the enzymes involved in bulk metabolic transforma- tions such as glycine N-methyltransferase and guanidinoac- etate methyltransferase. 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). GNMT, unlike most AdoMet- dependent methyltransferases, is an oligomeric protein consisting of four identical subunits (Ogawa & Fujioka, 1982a). The primary structure of the enzyme has been deduced from the nucleotide sequence of a cloned cDNA, and each subunit has been shown to contain 292 amino acid residues (Ogawa et al., 1987). Fujioka and his co-workers have characterized chemical and biological properties of the enzyme isolated from rat liver (Ogawa et al., 1987; Konishi & Fujioka, 1987, 1988). From enzymological and nutritional evidence, GNMT has been implicated as a major enzyme that regulates the AdoMet/AdoHcy ratio in some organisms (Ogawa & Fujio- ka, 1982a,b; Cook et al., 1989; Wagner et al., 1985; Balaghi et al., 1993). In addition to this role, GNMT has been shown to be a major folate binding protein of rat liver cytosol (Cook & Wagner, 1984) and, more recently, to be a polycyclic aromatic hydrocarbon binding protein (Raha et al., 1994) and a mediator of cytochrome P450 1A1 gene expression (Raha et al., 1995). To date, the crystal structures of only four AdoMet- dependent methyltransferases, catechol O-methyltransferase (COMT) (Vidgren et al., 1994), HhaI DNA methyltransferase (HhaI) (Cheng et al., 1993), TaqI DNA methyltransferase (TaqI) (Labahn et al., 1994), and HaeIII DNA methyltrans- ferase (HaeIII) (Reinisch et al., 1995), have been reported. These enzymes, despite having low degrees of sequence homology, show strikingly similar structures in the AdoMet binding domains, suggesting that methyltransferases in general have a common structure at the AdoMet binding site. Here we report the crystal structure of GNMT complexed ² The work carried out at the University of Kansas has been supported by NIH Grant GM37233 and in part by the Kansas Health Foundation and the Marion Merrell Dow Foundation. ‡ The atomic coordinates have been deposited with the Brookhaven Protein Data Bank (entry name 1XVA). * Authors to whom correspondence should be addressed. § University of Kansas. | Toyama Medical and Pharmaceutical University. X Abstract published in AdVance ACS Abstracts, September 1, 1996. 11985 Biochemistry 1996, 35, 11985-11993 S0006-2960(96)01068-9 CCC: $12.00 © 1996 American Chemical Society