Conformational Preferences of Pyridone Adenine Dinucleotides from Molecular Dynamics Simulations

Abstract

Pyridone adenine dinucleotides (ox-NADs) are redox inactive derivatives of the enzyme cofactor and substrate nicotinamide adenine dinucleotide (NAD) that have a carbonyl group at the C2, C4, or C6 positions of the nicotinamide ring. These aberrant cofactor analogs accumulate in cells under stress and are potential inhibitors of enzymes that use NAD(H). We studied the conformational landscape of ox-NADs in solution using molecular dynamics simulations. Compared to NAD+ and NADH, 2-ox-NAD and 4-ox-NAD have an enhanced propensity for adopting the anti conformation of the pyridone ribose group, whereas 6-ox-NAD exhibits greater syn potential. Consequently, 2-ox-NAD and 4-ox-NAD have increased preference for folding into compact conformations, whereas 6-ox-NAD is more extended. ox-NADs have distinctive preferences for the orientation of the pyridone amide group, which are driven by intramolecular hydrogen bonding and steric interactions. These conformational preferences are compared to those of protein-bound NAD(H). Our results may help in identifying enzymes targeted by ox-NADs.

Synthesis, Detection, and Metabolism of Pyridone Ribosides, Products of NAD Overoxidation

“…While each of the ox-NAD isomers was readily consumed in the presence of the liver extract as detected by 1 H NMR, we did not observe the appearance of the pyridone nucleobases, PY. Crucially, the location of the carbonyl on the pyridone moiety did not affect the rates at which the ox-NAD degraded to the PYR . However, over time, we observed that the signal to noise was lost, likely due to the precipitation of liver proteins binding to the pyridone metabolites.…”

“…Crucially, the location of the carbonyl on the pyridone moiety did not affect the rates at which the ox-NAD degraded to the PYR. 28 However, over time, we observed that the signal to noise was lost, likely due to the precipitation of liver proteins binding to the pyridone metabolites. To ensure that the enzymes responsible for the formation of PY species were not simply absent from the liver extract, we checked whether 2-, 4-, and 6-PYR could be substrates of the purine nucleoside phosphorylase, PNP, 31 or of the pyrimidine nucleoside phosphorylase, UPP1.…”

“…Each ox-NAD isomer is a mimic of NAD­(H) . One circumstance where the presence of ox-NAD might affect NAD­(H) biochemistry is when NAD­(H)-dependent redox enzymes are used to measure NAD­(H) levels in biological samples, such as the Promega NAD/NADH-Glo assay used to measure NAD in cells and tissue extracts.…”

“…Each ox-NAD isomer is a mimic of NAD(H). 28 One circumstance where the presence of ox-NAD might affect NAD(H) biochemistry is when NAD-(H)-dependent redox enzymes are used to measure NAD(H) levels in biological samples, such as the Promega NAD/ NADH-Glo assay used to measure NAD in cells and tissue extracts. The NAD/NADH-Glo assays use a two-enzyme system whereby a NAD to NADH cycling enzyme converts NAD to NADH, and a luciferase oxidizes NADH back to NAD while generating a luminescent product.…”

The Unexpected Functional Diversity of Photoexcited NAD

“…The explanation for the difference in populating long-lived excited states between NADH and NAD + might arise from the fact that a small amount (<15%) of NAD + molecules are in the folded form or nicotinamide could have poorer electron-donating properties than its reduced form. 10, 36 Hence, we hypothesize that any inter-ring excited-state electron transfer in NAD + may have very low quantum efficiency. To gain insight into the population of excited states of NADH and NAD + , we conducted an analysis of the GSB signal at 1623 cm -1 .…”