Nicotinamide N-methyltransferase catalyses the N-methylation of the endogenous β-carboline norharman: evidence for a novel detoxification pathway

Abstract

Nicotinamide N-methyltransferase (NNMT) is responsible for the N-methylation of nicotinamide to 1-methylnicotinamide. Our recent studies have demonstrated that NNMT regulates cellular processes fundamental to the correct functioning and survival of the cell. It has been proposed that NNMT may possess β-carboline (BC) N-methyltransferase activity, endogenously and exogenously produced pyridine-containing compounds which, when N-methylated, are potent inhibitors of Complex I and have been proposed to have a role in the pathogenesis of Parkinson's disease. We have investigated the ability of recombinant NNMT to N-methylate norharman (NH) to 2-N-methylnorharman (MeNH). In addition, we have investigated the toxicity of the BC NH, its precursor 1,2,3,4-tetrahydronorharman (THNH) and its N-methylated metabolite MeNH, using our in vitro SH-SY5Y NNMT expression model. Recombinant NNMT demonstrated NH 2N-methyltransferase activity, with a Km of 90 ± 20 µM, a kcat of 3 × 10(-4) ± 2 × 10(-5) s(-1) and a specificity constant (kcat/Km) of 3 ± 1 s(-1) M(-1) THNH was the least toxic of all three compounds investigated, whereas NH demonstrated the greatest, with no difference observed in terms of cell viability and cell death between NNMT-expressing and non-expressing cells. In NNMT-expressing cells, MeNH increased cell viability and cellular ATP concentration in a dose-dependent manner after 72 and 120 h incubation, an effect that was not observed after 24 h incubation or in non-NNNT-expressing cells at any time point. Taken together, these results suggest that NNMT may be a detoxification pathway for BCs such as NH.

Inhibitors of nicotinamide N-methyltransferase designed to mimic the methylation reaction transition state

“…Previous studies revealed norharmane to be a weak substrate for NNMT with a K M value of 90 µM, significantly lower than that of the native substrate NA (K M 200 µM), but with a very low turnover rate. 22 We were therefore interested in assessing whether such a strong binding, slowly converted NNMT substrate might in fact act as an inhibitor by occupying the NNMT active site. 16,22 Indeed, it was found that norharmane inhibits NNMT with an IC 50 of 115.3 ± 20.6 µM.…”

“…22 We were therefore interested in assessing whether such a strong binding, slowly converted NNMT substrate might in fact act as an inhibitor by occupying the NNMT active site. 16,22 Indeed, it was found that norharmane inhibits NNMT with an IC 50 of 115.3 ± 20.6 µM. The low K M is offset by a diminished turnover rate giving norharmane the ability to occupy the NNMT active site and effectively block the methylation of nicotinamide.…”

The kinetic analysis of the N -methylation of 4-phenylpyridine by nicotinamide N -methyltransferase: Evidence for a novel mechanism of substrate inhibition

“…These studies have been extensively cited in the literature to justify the identification of NNMT as responsible for the N-methylation of a range of pyridine-containing compounds such as quinolines and β-carbolines (Parsons et al, 2002(Parsons et al, , 2003Williams and Ramsden, 2005). Our recent studies have shown that the majority of these are poor NNMT substrates, with only isoquinoline demonstrating a catalytic efficiency (k cat /K m ) value greater than 30% of that observed for nicotinamide (van Haren et al, 2016;Thomas et al, 2016).…”

Photophysical and Photochemical Properties of Naturally Occurring normelinonine F and Melinonine F Alkaloids and Structurally Related N(2)‐ and/or N(9)‐methyl‐β‐carboline Derivatives

“…The unsubstituted 1,2,3,4‐tetrahydro‐ β Cs and the unsubstituted full‐aromatic nor harmane (9H‐pyrido[3,4‐b]indole; nHo) crosses the blood–brain barrier penetrating into the brain . Inside the brain, nHo is converted by certain S‐adenosyl methionine‐dependent N‐methyltransferases to the N‐methyl‐ β C cation named 2‐methyl‐ nor harmanium (2‐Me‐nHo) and subsequently to the 2,9‐dimethyl‐ nor harmanium cation (2,9‐diMe‐nHo) .A broad spectrum of biological activity has been reported for N‐methyl‐ β Cs (quaternary βCs): (1) They were described as potential pathogenetic factors in Parkinson's disease . The main mechanism through which these alkaloids would exert their effect still remains elusive.…”

“…The unsubstituted 1,2,3,4-tetrahydro-bCs and the unsubstituted full-aromatic norharmane (9H-pyrido [3,4-b]indole; nHo) crosses the bloodbrain barrier penetrating into the brain (21,22). Inside the brain, nHo is converted by certain S-adenosyl methionine-dependent N-methyltransferases to the N-methyl-bC cation named 2-methylnorharmanium (2-Me-nHo) and subsequently to the 2,9-dimethylnorharmanium cation (2,9-diMe-nHo) (23).…”