title: Structural basis of substrate recognition in human nicotinamide N-methyltransferase.
abstract: Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridines, and other analogues using S-adenosyl-l-methionine as donor. NNMT plays a significant role in the regulation of metabolic pathways and is expressed at markedly high levels in several kinds of cancers, presenting it as a potential molecular target for cancer therapy. We have determined the crystal structure of human NNMT as a ternary complex bound to both the demethylated donor S-adenosyl-l-homocysteine and the acceptor substrate nicotinamide, to 2.7 Å resolution. These studies reveal the structural basis for nicotinamide binding and highlight several residues in the active site which may play roles in nicotinamide recognition and NNMT catalysis. The functional importance of these residues was probed by mutagenesis. Of three residues near the nicotinamide's amide group, substitution of S201 and S213 had no effect on enzyme activity while replacement of D197 dramatically decreased activity. Substitutions of Y20, whose side chain hydroxyl interacts with both the nicotinamide aromatic ring and AdoHcy carboxylate, also compromised activity. Enzyme kinetics analysis revealed k(cat)/K(m) decreases of 2-3 orders of magnitude for the D197A and Y20A mutants, confirming the functional importance of these active site residues. The mutants exhibited substantially increased K(m) for both NCA and AdoMet and modestly decreased k(cat). MD simulations revealed long-range conformational effects which provide an explanation for the large increase in K(m)(AdoMet) for the D197A mutant, which interacts directly only with nicotinamide in the ternary complex crystal structure.
authors.affiliation: Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, United States.
authors.forename: Yi
authors.initials: Y
authors.lastname: Peng
authors.forename: Davide
authors.initials: D
authors.lastname: Sartini
authors.forename: Valentina
authors.initials: V
authors.lastname: Pozzi
authors.forename: Dennis
authors.initials: D
authors.lastname: Wilk
authors.forename: Monica
authors.initials: M
authors.lastname: Emanuelli
authors.forename: Vivien C
authors.initials: VC
authors.lastname: Yee
mesh_terms: D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009536:Niacinamide / Q000737:chemistry / Q000378:metabolism; D050903:Nicotinamide N-Methyltransferase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D012436:S-Adenosylmethionine / Q000737:chemistry*; D013329:Structure-Activity Relationship
chemical_list: D009536:Niacinamide; D012436:S-Adenosylmethionine; C494406:NNMT protein, human; D050903:Nicotinamide N-Methyltransferase