19 research outputs found
A Family of Water Immiscible, Dipolar Aprotic, Diamide Solvents from Succinic Acid
Three dipolar aprotic solvents were designed to possess high dipolarity and low toxicity: N , N , N ', N '-tetrabutylsuccindiamide (TBSA), N , N '-diethyl- N , N '-dibutylsuccindiamide (EBSA), N , N '-dimethyl- N , N '-dibutylsuccindiamide (MBSA). They were synthesized catalytically using a K60 silica catalyst in a solventless system. Their water-immiscibility stands out as an unusual and useful property for dipolar aprotic solvents. They were tested in a model Heck reaction, metal-organic framework syntheses, and a selection of polymer solubility experiments where their performances were found to be comparable to traditional solvents. Furthermore, MBSA was found to be suitable for the production of an industrially-relevant membrane from polyethersulphone. An integrated approach involving in silico analysis based on available experimental information, prediction model outcomes and read across data, as well as a panel of in vitro reporter gene assays covering a broad range of toxicological endpoints was used to assess toxicity. These in silico and in vitro tests suggested no alarming indications of toxicity in the new solvents
Free energies of binding of R- and S-propranolol to wild-type and F483A mutant cytochrome P450 2D6 from molecular dynamics simulations
Detailed molecular dynamics (MD) simulations have been performed to reproduce and rationalize the experimental finding that the F483A mutant of CYP2D6 has lower affinity for R-propranolol than for S-propranolol. Wild-type (WT) CYP2D6 does not show this stereospecificity. Four different approaches to calculate the free energy differences have been investigated and were compared to the experimental binding data. From the differences between calculations based on forward and backward processes and the closure of thermodynamic cycles, it was clear that not all simulations converged sufficiently. The approach that calculates the free energies of exchanging R-propranolol with S-propranolol in the F483A mutant relative to the exchange free energy in WT CYP2D6 accurately reproduced the experimental binding data. Careful inspection of the end-points of the MD simulations involved in this approach, allowed for a molecular interpretation of the observed differences
Advances in Understanding Xenobiotic Metabolism.
The understanding of how exogenous chemicals (xenobiotics) are metabolized, distributed, and eliminated is critical to determine the impact of the chemical and its metabolites to the (human) organism. This is part of the research and educational discipline ADMET (absorption, distribution, metabolism, elimination, and toxicity). Here, we review the work of Jan Commandeur and colleagues who have not only made a significant impact in understanding of phase I and phase II metabolism of several important compounds but also contributed greatly to the development of experimental techniques for the study of xenobiotic metabolism. Jan Commandeur's work has covered a broad area of research, such as the development of online screening methodologies, the use of a combination of enzyme mutagenesis and molecular modeling for structure-activity relationship (SAR) studies, and the development of novel probe substrates. This work is the bedrock of current activities and brings the field closer to personalized (cohort-based) pharmacology, toxicology, and hazard/risk assessment.publishersversionpublishe
EPR-monitored redox titration curves of FumA (•, red) and FumB (□, blue).
<p>The two points at low, undefined potential represent samples reduced with excess dithionite (10 mM). The solid lines represents fits to the Nernst equation: . Fit parameters for FumA: E<sub>m</sub> = −300±6 mV; and for FumB: E<sub>m</sub> = −283±9 mV.</p
Amino acid sequence alignment of <i>E. coli</i> FumA and FumB. FumA and FumB share 90% sequence identity and 95% sequence similarity.
<p>*Cysteine residues strictly conserved in multiple sequence alignment, using the ‘Cobalt’ program, from the 500 homologs exhibiting >72% sequence identity with <i>E. coli</i> FumA.</p
Biochemical Similarities and Differences between the Catalytic [4Fe-4S] Cluster Containing Fumarases FumA and FumB from <em>Escherichia coli</em>
<div><h3>Background</h3><p>The highly homologous [4Fe-4S] containing fumarases FumA and FumB, sharing 90% amino acid sequence identity, from <em>Escherichia coli</em> are differentially regulated, which suggests a difference in their physiological function. The ratio of FumB over FumA expression levels increases by one to two orders of magnitude upon change from aerobic to anaerobic growth conditions.</p> <h3>Methodology/Principal Findings</h3><p>To understand this difference in terms of structure-function relations, catalytic and thermodynamic properties were determined for the two enzymes obtained from homologous overexpression systems. FumA and FumB are essentially identical in their Michaelis-Menten kinetics of the reversible fumarate to L-malate conversion; however, FumB has a significantly greater catalytic efficiency for the conversion of D-tartrate to oxaloacetate consistent with the requirement of the <em>fumB</em> gene for growth on D-tartrate. Reduction potentials of the [4Fe-4S]<sup>2+</sup> Lewis acid active centre were determined in mediated bulk titrations in the presence of added substrate and were found to be approximately −290 mV for both FumA and FumB.</p> <h3>Conclusions/Significance</h3><p>This study contradicts previously published claims that FumA and FumB exhibit different catalytic preferences for the natural substrates L-malate and fumarate. FumA and FumB differ significantly only in the catalytic efficiency for the conversion of D-tartrate, a supposedly non-natural substrate. The reduction potential of the substrate-bound [4Fe-4S] active centre is, contrary to previously reported values, close to the cellular redox potential.</p> </div
EPR spectra of FumA (red) and FumB (blue).
<p>a FumA [3Fe-4S]<sup>1+</sup> clusters; as isolated, not regenerated. b FumB [3Fe-4S]<sup>1+</sup> clusters; as isolated, not regenerated. c FumA [4Fe-4S]<sup>1+</sup> clusters; regenerated, reduced and in the presence of 5 mM fumarate. d FumB [4Fe-4S]<sup>1+</sup> clusters; regenerated, reduced and in the presence of 5 mM fumarate. The g-values are 2.032, 1.914 and 1.822 for FumA and 2.032, 1.916 and 1.821 for FumB. EPR parameters: microwave frequency a 9.630 GHz, b 9.631 GHz, c 9.407 GHz, d 9.631 GHz; microwave power a 8.0 mW, b 8.0 mW, c 20 mW, d 20 mW; modulation frequency 100 kHz; modulation amplitude a 0.63 mT, b 0.63 mT, c 1.25 mT, d 1.25 mT; temperature a 14.5 K, b 14.5 K, c 16K, d 14.5 K.</p
Oxygen sensitivity of FumA (•, red) and FumB (□, blue).
<p>Fumarase activity was measured after air oxidation for 0, 0.5, 1 or 2 minutes. The residual activity was plotted as a percentage of the initial activity. The solid lines represent fits to the following equation:. The fit parameters were as follows, for FumA: <i>A</i> = 5±6%; <i>k<sub>inact</sub></i> = (1.8±0.5)·10<sup>2</sup> M<sup>−1</sup>s<sup>−1</sup>, for FumB: <i>A</i> = 6±6%; <i>k<sub>inact</sub></i> = (1.6±0.4)·10<sup>2</sup> M<sup>−1</sup>s<sup>−1</sup>.</p
Kinetic parameters of fumarase and tartrate dehydratase activity of FumA and FumB<sup>a</sup>.
a<p>All values are corrected for Fe-S cluster content.</p>b<p><i>K<sub>m</sub></i> in mM.</p>c<p><i>V<sub>max</sub></i> in µmol product/minute/mg enzyme.</p>d<p><i>k<sub>cat</sub>/K<sub>m</sub></i> in s<sup>−1</sup> M<sup>−1</sup>.</p>e<p><i>K<sub>eq</sub></i> is the equilibrium constant for the hydration of fumarate as calculated using the Haldane relationship: <i>K<sub>eq</sub></i> = <i>(k<sub>cat</sub>/K<sub>M</sub>)<sub>fumarate</sub>/(k<sub>cat</sub>/K<sub>M</sub>)<sub>L-malate</sub></i>.</p