Noncoupled Fluorescent Assay for Direct Real-Time Monitoring of Nicotinamide N-Methyltransferase Activity

Biochemistry. 2017 Feb 14;56(6):824-832. doi: 10.1021/acs.biochem.6b01215. Epub 2017 Jan 30.

Abstract

Nicotinamide N-methyltransferase (NNMT) is an important biotransforming enzyme that catalyzes the transfer of a labile methyl group from the ubiquitous cofactor S-5'-adenosyl-l-methionine (SAM) to endogenous and exogenous small molecules to form methylated end products. NNMT has been implicated in a number of chronic disease conditions, including metabolic disorders, cardiovascular disease, cancer, osteoarthritis, kidney disease, and Parkinson's disease. We have developed a novel noncoupled fluorescence-based methyltransferase assay that allows direct ultrasensitive real-time detection of the NNMT reaction product 1-methylquinolinium. This is the first assay reported to date to utilize fluorescence spectroscopy to directly monitor NNMT product formation and activity in real time. This assay provided accurate kinetic data that allowed detailed comparative analysis of the NNMT reaction mechanism and kinetic parameters. A reaction model based on a random bireactant mechanism produced global curve fits that were most consistent with steady-state initial velocity data collected across an array of substrate concentrations. On the basis of the reaction mechanism, each substrate could independently bind to the NNMT apoenzyme; however, both substrates bound to the complementary binary complexes with an affinity ∼20-fold stronger compared to their binding to the apoenzyme. This reaction mechanism implies either substrate-induced conformational changes or bireactant intermolecular interactions may stabilize the binding of the substrate to the binary complex and formation of the ternary complex. Importantly, this assay could rapidly generate concentration response curves for known NNMT inhibitors, suggesting its applicability for high-throughput screening of chemical libraries to identify novel NNMT inhibitors. Furthermore, our novel assay potentially offers a robust detection technology for use in SAM substrate competition assays for the discovery and development of SAM-dependent methyltransferase inhibitors.

Publication types

  • Comparative Study

MeSH terms

  • Apoenzymes / antagonists & inhibitors
  • Apoenzymes / chemistry
  • Apoenzymes / genetics
  • Apoenzymes / metabolism
  • Biocatalysis / drug effects
  • Calibration
  • Enzyme Inhibitors / pharmacology
  • High-Throughput Screening Assays
  • Humans
  • Limit of Detection
  • Methylation / drug effects
  • Models, Molecular*
  • Nicotinamide N-Methyltransferase / antagonists & inhibitors
  • Nicotinamide N-Methyltransferase / chemistry
  • Nicotinamide N-Methyltransferase / genetics
  • Nicotinamide N-Methyltransferase / metabolism*
  • Protein Conformation
  • Protein Refolding / drug effects
  • Quinolinium Compounds / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Reproducibility of Results
  • S-Adenosylmethionine / metabolism
  • Spectrometry, Fluorescence

Substances

  • Apoenzymes
  • Enzyme Inhibitors
  • Quinolinium Compounds
  • Recombinant Proteins
  • 1-methylquinolinium
  • S-Adenosylmethionine
  • NNMT protein, human
  • Nicotinamide N-Methyltransferase