Spectroscopic and computational studies on the adenosylcobalamin-dependent methylmalonyl-CoA mutase: evaluation of enzymatic contributions to Co-C bond activation in the Co3+ ground state

Methylmalonyl-CoA mutase (MMCM) is an enzyme that utilizes the adenosylcobalamin (AdoCbl) cofactor to catalyze the rearrangement of methylmalonyl-CoA to succinyl-CoA. Despite many years of dedicated research, the mechanism by which MMCM and related AdoCbl-dependent enzymes accelerate the rate for homolytic cleavage of the cofactor's Co-C bond by approximately 12 orders of magnitude while avoiding potentially harmful side reactions remains one of the greatest subjects of debate among B(12) researchers. In this study, we have employed electronic absorption (Abs) and magnetic circular dichroism (MCD) spectroscopic techniques to probe cofactor/enzyme active site interactions in the Co(3+)Cbl "ground" state for MMCM reconstituted with both the native cofactor AdoCbl and its derivative methylcobalamin (MeCbl). In both cases, Abs and MCD spectra of the free and enzyme-bound cofactor are very similar, indicating that replacement of the intramolecular base 5,6-dimethylbenzimidazole (DMB) by a histidine residue from the enzyme active site has insignificant effects on the cofactor's electronic properties. Likewise, spectral perturbations associated with substrate (analogue) binding to holo-MMCM are minor, arguing against substrate-induced enzymatic Co-C bond activation. As compared to the AdoCbl data, however, Abs and MCD spectral changes for the sterically less constrained MeCbl cofactor upon binding to MMCM and treatment of holoenzyme with substrate (analogues) are much more substantial. Analysis of these changes within the framework of time-dependent density functional theory calculations provides uniquely detailed insight into the structural distortions imposed on the cofactor as the enzyme progresses through the reaction cycle. Together, our results indicate that, although the enzyme may serve to activate the cofactor in its Co(3+)Cbl ground state to a small degree, the dominant contribution to the enzymatic Co-C bond activation presumably comes through stabilization of the Co(2+)Cbl/Ado. post-homolysis products.     

Buffers for the Physiological pH Range: Thermodynamics of the Second Dissociation of N-(2-Hydroxyethyl)piperazine-N′-2-hydroxypropanesulfonic Acid From 5 to 55°C

The second acidic dissociation constants pK ₂ of the ampholyte N-(2-hydroxyethyl) piperazine-N-2-hydroxypropanesulfonic acid (HEPPSO) have been determined at seven temperatures from 5 to 55°C from emf measurements utilizing hydrogen and silver–silver chloride cells without liquid junction. The thermodynamic quantities, G°, H°,S°, and C _p ^o have been calculated from the temperature coefficient of pK ₂. At 25°C, the pK ₂ = 8.042 and at 37°C, pK ₂ = 7.876; hence, buffer solutions of HEPPSO and NaHEPPSOate are important for pH control in the region close to that of clinical fluids (blood serum). Conventional pH values from 5 to 55°C as well as those obtained from liquid junction correction at 25 and 37°C have been reported for three buffer solutions with the compositions (molality scale): (1) equimolal mixture of HEPPSO (0.04 m) + NaHEPPSOate (0.04 m) + NaCl (0.12 m); (2) HEPPSO (0.08 m) + NaHEPPSOate (0.08 m); and (3) HEPPSO (0.08 m) + NaHEPPSOate (0.08 m) + NaCl (0.08 m).     

The synthesis of dehydrotryptophan and dehydrotryptophan-containing peptides

Dehydrotryptophan and its derivatives are non-proteinogenic amino acids commonly found in peptide-based natural products produced by microorganisms, marine organisms and plants. These non-proteinogenic amino acids are found in secondary metabolites and are formed by post translational modification processes. Although comprehensive reviews on the synthesis of dehydroamino acids are available, this perspective focuses solely on methods to synthesise the dehydrotryptophan-containing segment of naturally occurring peptides, amino acids and their derivatives.     

Bioorthogonal Probes for the Study of MDM2‐p53 Inhibitors in Cells and Development of High‐Content Screening Assays for Drug Discovery

To study the behavior of MDM2‐p53 inhibitors in a disease‐relevant cellular model, we have developed and validated a set of bioorthogonal probes that can be fluorescently labeled in cells and used in high‐content screening assays. By using automated image analysis with single‐cell resolution, we could visualize the intracellular target binding of compounds by co‐localization and quantify target upregulation upon MDM2‐p53 inhibition in an osteosarcoma model. Additionally, we developed a high‐throughput assay to quantify target occupancy of non‐tagged MDM2‐p53 inhibitors by competition and to identify novel chemical matter. This approach could be expanded to other targets for lead discovery applications.     

Behaviour of polycyclic aromatic hydrocarbons in dissolved,colloidal, and particulate phases in sedimentary cores

Solid-phase microextraction (SPME) has been successfully used for extracting polycyclic aromatic hydrocarbons (PAHs) from porewater samples from the Mersey Estuary, UK. The majority of the PAHs in porewater samples are associated with colloids due to the high DOC concentrations. The truly dissolved PAH concentrations varied from 66 to 1050?ng?L^?1 in core 2 and from 95 to 740?ng?L^?1 in core 3, and were dominated by naphthalene, fluoranthene, and pyrene. Although absent in the dissolved phase, the high-molecular-mass compounds were found in the colloid-associated fraction of porewater. PAHs in sediments arose from a range of compounds with 4- and 5-ring PAHs dominating. The partitioning of PAHs between sediment and porewater shows that PAHs are enriched in the sediment phase. When the soot carbon content was considered, predictions of the partition behaviour were found to agree more closely with the observed distribution. The results reiterate the importance of evaluating the speciation of organic pollutants in both porewater and sediments in order to accurately predict their environmental fate and potential toxicity.     

The effect of photomask resolution on separation efficiency on microfabricated devices

Separation quality on glass microfluidic devices fabricated from photomasks of different optical resolutions was compared by measuring the dispersion (apparent diffusion) coefficients of a set of standard compounds separated on these devices. Currently, the channel manifolds of most microfluidic devices are patterned using chrome photomasks. A much cheaper, more robust alternative to chrome photomasks are laser photoplotted masks. The primary disadvantage to using laser photoplots is that the optical resolution of these masks is not as high as that of chrome masks, and this feature increases the side-wall roughness of etched channel manifolds patterned using such masks. The increased wall roughness may affect the fluid flow within the channels and, therefore, the separation quality. To determine the effect of increased sidewall channel roughness, microchip channel manifolds were patterned in soda lime glass using a chrome photomask and laser photoplots printed at resolutions of 620, 1240, 3100 and 6200 dots per centimetre (dpc). Separations were performed on these devices using dilute solutions of fluorescently labeled amino acids. The peak variances of the amino acids were calculated at increasing distances down the separation channel and plotted as a function of migration time. From this plot, dispersion coefficients of the analytes were measured. This allowed for a reliable, relatively easy, direct separation analysis among microchips fabricated from the various photomasks. After multiple separations using microchips fabricated from each resolution mask, we found that the change in sidewall surface roughness did not significantly affect the dispersion coefficients measured, and thus the separation quality. The lower mask resolution limit, rather, was governed by the fidelity to which the mask could capture the original CAD design.     

Phenanthrene removal in unsaturated soils treated by electrokinetics with different surfactants—Triton X-100 and rhamnolipid

In this study, the remediation performance of electrokinetic (EK) technology integrated with different surfactants for removing phenanthrene from unsaturated soils was investigated. A synthetic surfactant (Triton X-100) and a biosurfactant (rhamnolipid) were used to enhance phenanthrene solubility and removal efficiency during EK process. Results indicate that the electro-osmotic flow (EOF) rate in the rhamnolipid system is higher than that in Triton X-100. Using the EK technology for the removal of phenanthrene in the presence of rhamnolipid was more efficient than in the presence of Triton X-100. In addition to the transport mechanism of phenanthrene in EK system, the presence of rhamnolipid may promote microbial growth in the soil–water system and bring about biodegradation of phenanthrene. A diffusion–advection–sorption (DAS) model was solved by MATLAB, based on the linear sorption isotherm at the non-equilibrium condition, which is feasible to simulate the movement of phenanthrene during the EK + Triton X-100 treatment.