Structural Basis of Substrate Recognition in Human Nicotinamide N-Methyltransferase

Abstract

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridines and other analogs 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 2.7Å 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 kcat/Km 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 Km for both NCA and AdoMet and modestly decreased kcat. MD simulations revealed long-range conformational effects which provide an explanation for the large increase in Km(AdoMet) for the D197A mutant, which interacts directly only with nicotinamide in the ternary complex crystal structure.

Identification and Characterization of Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma Cell Lines

“…In the present work, we also focused on the expression of the enzyme Nicotinamide N-Methyltransferase, which catalyzed the N-methylation of nicotinamide, pyridines, and other structural analogs, playing a fundamental role in the biotransformation of many xenobiotics [45,46]. An enhanced expression of NNMT has been reported in a number of cancers, such as glioblastoma [47], stomach adenocarcinoma [48,49], papillary thyroid cancers [50,51], renal [52,53], and oral squamous cell carcinomas [54][55][56], colorectal cancer [57], bladder [58], lung and pancreatic cancers [59][60][61].…”

SerumN1-Methylnicotinamide Is Associated With Obesity and Diabetes in Chinese

“…NNMT is activated by its substrate, nicotinamide (26). Nicotinamide overload induces higher plasma me-NAM levels in individuals with T2DM than in healthy control subjects (15).…”

The m1A58 modification in eubacterial tRNA: An overview of tRNA recognition and mechanism of catalysis by TrmI

“…In addition to the proximity and orientation requirements, the enzyme could be involved in the stabilization of the transition state of the reaction, three parameters that overall increase the rate of methyl-transfer [52]. Even though the mechanism of m 1 A methylation has not been thoroughly investigated on its own, some information can be inferred from the study of the N-methylation mechanism of other compounds such as nicotinamide [54] or intermediate products of the caffeine pathway [55,56]. In these methylation reactions, the endocyclic nitrogens are sometimes initially protonated (e.g.…”