Binding Affinity Studies of Nicotinamide N-methyltransferase and Ligands by Saturation Transfer Difference NMR

Abstract

Introduction: Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide with S-adenosine-L-methionine (SAM) as the methyl donor. Abnormal expression of NNMT is associated with many diseases (such as multiple cancers and metabolic and liver diseases), making NNMT a potential therapeutic target. Limited studies concerning the enzyme-substrate/inhibitor interactions could be found to fully understand the detailed reaction mechanism. Methods: The binding affinity and ligand binding epitopes of nicotinamide or SAH for binding NNMT and its mutants were determined using saturated transfer difference (STD) nuclear magnetic resonance (NMR) techniques combined with site-directed mutagenesis. Results: The average dissociation constant of WT NNMT with nicotinamide and S-adenosine homocysteine (SAH) was 5.5 ± 0.9 mM and 1.2 ± 0.3 mM, respectively, while the mutants Y20F and Y20G with nicotinamide were up to nearly 4 times and 20 times that of WT and with SAH nearly 2 times and 5 times that of WT. The data suggested that WT had the highest binding affinity for nicotinamide or SAH, followed by Y20F and Y20G, which was consistent with its catalytic activity. Conclusion: The binding affinity of nicotinamide and SAH to NNMT and its mutants were obtained by STD NMR in this study. It was found that nicotinamide and SAH bind to WT in a particular orientation, and Y20 is critical for their binding orientation and affinity to NNMT.