Nuclear magnetic resonance based structure of the protoberberine alkaloid coralyne and its self-association by spectroscopy techniques

Coralyne is an important alkaloid due to its anti-cancer and other medicinal properties.It targets DNA in cells and acts as human topoisomerase-I poison,telomerase inhibitor and nucleic acid intercalator.It has high tendency to undergo self-association,which is a matter of concern for therapeutic applications.The understanding of its interaction with DNA requires precise knowledge of chemical shifts in Nuclear Magnetic Resonance(NMR)spectra besides self-association.The present study is the first report of a complete assignment of all 1H/13C resonances in NMR spectra of coralyne in DMSO-d6 using one dimensional 1H/13C and two dimensional NMR experiments.The chemical shift of all proton and several 13C resonances have also been obtained in D2O and ethanol-d6.The same has been calculated using Density Functional Theory(DFT).NMR spectra of coralyne show upfield shift of 0.6e1.2 ppm in aromatic ring protons suggesting stacking interactions.Apart from 11 intra molecular NOE cross peaks in 2D 1H e1H ROESY spectra,3 short distance NOE correlations,H6e10OCH3,H5e10OCH3 and H12e16CH3,give direct independent evidence of the formation of a stacked dimer.The absorbance,fluorescence,circular dichroism and fluorescence lifetime experiments conducted in the present investigations corroborate results obtained by NMR.

The Study of Carbamoyl Phosphate Synthetase 1 Deficiency Sheds Light on the Mechanism for Switching On/Off the Urea Cycle

Carbamoyl phosphate synthetase i （CPS1） deficiency （CPS1D） is an inborn error of the urea cycle having autosomal （2q34） recessive inheritance that can cause hyperammonemia and neonatal death or mental retardation. We analyzed the effects on CPS1 activity, kinetic parameters and enzyme stability of missense mutations reported in patients with CPS1 deficiency that map in the 20-kDa C-terminal domain of the enzyme. This domain turns on or off the enzyme depending on whether the essential allosteric activator of CPS 1, N-acetyl- L-glutamate （NAG）, is bound or is not bound to it. To carry out the present studies, we exploited a novel system that allows the expression in vitro and the purification of human CPS1, thus permitting site-directed mutagenesis. These studies have clarified disease causation by individual mutations, identifying functionally important residues, and revealing that a number of mutations decrease the affinity of the enzyme for NAG. Patients with NAG affinity-decreasing mutations might benefit from NAG site saturation therapy with N-carbamyl-L- glutamate （a registered drug, the analog of NAG）. Our results, together with additional present and prior site-directed mutagenesis data for other residues mapping in this domain, suggest an NAG-triggered conformational change in the 134-~4 loop of the C-terminal domain of this enzyme. This change might be an early event in the NAG activation process. Molecular dynamics simulations that were restrained according to the observed effects of the mutations are consistent with this hypothesis, providing further backing for this structurally plausible signaling mechanism by which NAG could trigger urea cycle activation via CPS1.