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

Harshini Neelakantan, Virginia Vance, Hua Yu Leo Wang, Stanton F. McHardy, Stanley Watowich

Research output: Contribution to journalArticle

11 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)824-832
Number of pages9
JournalBiochemistry
Volume56
Issue number6
DOIs
StatePublished - Feb 14 2017

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Nicotinamide N-Methyltransferase
Assays
Monitoring
Substrates
Methionine
Apoenzymes
Methyltransferases
Small Molecule Libraries
Kidney Neoplasms
Fluorescence Spectrometry
Fluorescence spectroscopy
Kidney Diseases
Reaction products
Kinetic parameters
Osteoarthritis
Parkinson Disease
Screening
Chronic Disease
Cardiovascular Diseases
Fluorescence

ASJC Scopus subject areas

  • Biochemistry

Cite this

Noncoupled Fluorescent Assay for Direct Real-Time Monitoring of Nicotinamide N-Methyltransferase Activity. / Neelakantan, Harshini; Vance, Virginia; Wang, Hua Yu Leo; McHardy, Stanton F.; Watowich, Stanley.

In: Biochemistry, Vol. 56, No. 6, 14.02.2017, p. 824-832.

Research output: Contribution to journalArticle

Neelakantan, Harshini ; Vance, Virginia ; Wang, Hua Yu Leo ; McHardy, Stanton F. ; Watowich, Stanley. / Noncoupled Fluorescent Assay for Direct Real-Time Monitoring of Nicotinamide N-Methyltransferase Activity. In: Biochemistry. 2017 ; Vol. 56, No. 6. pp. 824-832.
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