Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide pyridines and additional analogs using S-adenosyl-L-methionine as donor. 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 MK-0822 residues near the nicotinamide’s amide group substitution of S201 and Serpinf1 S213 had no effect on enzyme activity while replacement of D197 dramatically decreased activity. Substitutions of Y20 whose side chain hydroxyl interacts with both nicotinamide aromatic band and AdoHcy carboxylate also jeopardized activity. Enzyme kinetics evaluation revealed kcat/Km reduces of 2-3 purchases of magnitude for the D197A and Y20A mutants confirming the practical need for these energetic site residues. The mutants exhibited substantially increased Km for both AdoMet and NCA and modestly decreased kcat. MD simulations exposed long-range conformational results which offer an description for the top upsurge in Km(AdoMet) for the D197A mutant which interacts straight just with nicotinamide in the ternary complicated crystal framework. Nicotinamide (NCA) is a compound essential for the formation of NAD(H) and NADP(H) which are involved in many important biological processes including energy production cellular resistance to stress and longevity. Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme that catalyzes the N-methylation of NCA pyridines and analogs using S-adenosyl-L-methionine (AdoMet) as the methyl donor (1). NNMT plays a role in NCA metabolism and in the detoxification of many xenobiotics. The enzyme was first characterized in liver (2) where it is mainly expressed; there its activity varies 5-fold among individuals and has a bimodal frequency distribution which raises the possibility that a genetic polymorphism may be associated with its enzyme activity (1). In addition in Parkinson’s disease an enhanced NNMT activity seems to produce toxic N-methylpyridinium compounds advanced as possible neurotoxins underlying nigrostriatal degeneration (3 4 Lower expression levels of NNMT are also found in kidney lung placenta heart brain and muscle. Interestingly abnormally high expression of NNMT has been identified in various cancers such as papillary thyroid carcinoma colorectal cancer lung cancer and oral carcinoma (5-9). To elucidate the structural basis of NNMT catalysis and function and to aid the design of new NNMT inhibitors that may have therapeutic use crystal structures of NNMT bound to substrate or inhibitor are desirable. Although the crystal structures of human and mouse NNMT (hNNMT and mNNMT respectively) bound to the demethylated product S-adenosyl-L-homocysteine (AdoHcy) have been determined (unpublished MK-0822 PDB accession codes 2IIP and 2I62 respectively) they did not reveal the NCA acceptor substrate binding site. We report here the crystal structure of hNNMT bound to both AdoHcy and NCA. The structure reveals the protein features important for NCA binding and highlights several residues in MK-0822 the active site which were subsequently tested by mutagenesis for their possible importance in NCA recognition and NNMT catalysis. MATERIALS AND METHODS Cloning Expression and Purification Total RNA was isolated from normal renal tissue using the SV Total RNA Isolation System (Promega). RNA (1 μg) was reverse transcribed with a first strand cDNA synthesis kit II (Bio Basic) using oligo-dT18 primers. 0.5 μl of the reaction mixture was then subjected to PCR with Taq polymerase (total volume 25 μl) using the primers 5′-tcacatatggaatcaggcttca-3′ (forward) and 5′-ctaaagctttcacaggggtctg-3′ (reverse) to amplify the human NNMT ORF and to insert NdeI and HindIII restriction sites. The amplified and digested PCR product was cloned into a pT7-7 plasmid vector to obtain the expression construct pT7-7-wt-hNNMT used to transform BL21 (DE3) cells that were grown at 37°C to an OD600 of 1 1.0 before induction with 1mM IPTG and incubation overnight. Cells were harvested by centrifugation and resuspended in lysis buffer (10 mM Tris-HCl pH 8.6 PMSF 1 mM DTT 1 mM and aprotinin 2 μg/ml) before sonication and centrifugation. The supernatant was loaded onto a hydroxyapatite column equilibrated with 10 mM sodium phosphate pH 7.5 DTT 1 mM and purified wt-hNNMT eluted with a linear gradient of NaCl from 0 to 1 1 M in the MK-0822 equilibration buffer. Extensive crystallization trials failed to yield.