Solvent inhibition profiles and inverse solvent isotope effects for enzymatic methyl transfer catalyzed by nicotinamideN‐methyltransferase

Abstract

Nicotinamide N‐methyltransferase (NNMT) catalyzes the methyl transfer from universal methyl donor S‐adenosyl methionine (SAM) to nicotinamide (NA) to form the N‐methylnicotinamide. This important process is related to the level of nicotinamide adenine dinucleotide (NAD+) in vivo; thus, NNMT is regarded as an important drug target linked with various diseases. Although NNMT has been extensively studied in the area of biology and medicine, the detailed mechanism of NNMT is still not clear, especially in the aspect of the role of solvents (water) in the enzyme catalysis. Here, we have examined the effects of three common organic solvents (dimethyl sulfoxide [DMSO], methanol, and acetonitrile) and deuterated water to explore the enzymatic methyl transfer activity by NNMT (wild type [WT]) and its mutants. Competitive inhibition was detected for DMSO and acetonitrile as the solvent, and a subtle inhibitory effect was observed with methanol. Molecular docking suggested DMSO and acetonitrile compete with both cofactor and substrate. However, methanol is mainly bound to compete for the substrate. Furthermore, the activity increased when deuterated water was substituted for water with an inverse solvent isotope effect D2Okcat/Km = 0.49 ± 0.17 for WT and D2Okcat/Km = 0.32 ± 0.08 for Y20F. These effects in this enzymatic methyl transfer system without any metal ion or acid/base catalysis were due to the stabilization of protein provided by deuterated water.

Automated High-Throughput System Combining Small-Scale Synthesis with Bioassays and Reaction Screening

“…83–85,106,107 Therefore, the development of high-throughput assays of NNMT activity is increasingly relevant to biological understanding and therapeutics. Traditional NNMT high-throughput assays rely on radioactive labeling (typically radiolabeled SAM) or fluorescence generation through coupled chemical or enzymatic reactions, 82 such as the 2,7-naphthyridin-1( 7H )-one formation between N -methylnicotinamide and acetylbenzofurane, 108,109 or the enzymatic conversion of SAH into homocysteine, which can be detected using thiol-specific fluorescent probes. 110 These assays, despite being widely used and even commercially available, have several disadvantages, including the use of radioactive material or labels and the need for multiple preparation steps, which makes them slow and labor-intensive.…”

“…84,85 Previous studies have explored several pyridines, quinolines, and cyclic amines, 85,113 finding, for instance, that quinoline can be a substrate of NNMT, 82 albeit with lower kinetic efficiency than pyridine. 85,109 However, the substrate scope tested has been modest (<30 compounds), yielding few trends in its description. Therefore, this is a challenging case in which label-free HTS would be greatly beneficial.…”