Determination Of Dissociation Constants Research Articles

Determination of acid dissociation constants and reaction kinetics of dimethylamine-based PPCPs with O3, NaClO, ClO2 and KMnO4

Dimethylamine-based pharmaceutical personal care products (DMA-based PPCPs) are a group of N-nitrosodimethylamine (NDMA) precursors. The acid dissociation constant (pKa) values of four DMA-based PPCPs were determined by potentiometric titration over the pH range of 3–11. The pKa values of ranitidine, nizatidine, doxylamine and carbinoxamine corresponding to the DMA moiety were 8.4, 6.8, 9.4 and 9.1, respectively. Competition reaction kinetics and pseudo-first-order reaction kinetics were used to determine the reaction rate constant (k) of the DMA-based PPCPs with O3, NaClO, ClO2 and KMnO4. Comparing the degradation rate constants of the four DMA-based PPCPs, the results of ClO2 oxidation were close, and for the other three oxidants, the order was kranitidine ≈ knizatidine > kdoxylamine ≈ kcarbinoxamine. Comparing the reaction rate of the four oxidants, for ranitidine and nizatidine, the order was kNaClO > kO3 > kKMnO4 > kClO2, and for doxylamine and carbinoxamine, the order was kO3 > kNaClO > kClO2 > kKMnO4.

Mass spectrometry-based analysis of macromolecular complexes of Staphylococcus aureus uracil-DNA glycosylase and its inhibitor reveals specific variations due to naturally occurring mutations.

The base excision repair pathway plays an important role in correcting damage induced by either physiological or external effects. This repair pathway removes incorrect bases from the DNA. The uracil base is among the most frequently occurring erroneous bases in DNA, and is cut out from the phosphodiester backbone via the catalytic action of uracil‐DNA glycosylase. Uracil excision repair is an evolutionarily highly conserved pathway and can be specifically inhibited by a protein inhibitor of uracil‐DNA glycosylase. Interestingly, both uracil‐DNA glycosylase (Staphylococcus aureus uracil‐DNA glycosylase; SAUDG) and its inhibitor (S. aureus uracil‐DNA glycosylase inhibitor; SAUGI) are present in the staphylococcal cell. The interaction of these two proteins effectively decreases the efficiency of uracil‐DNA excision repair. The physiological relevance of this complexation has not yet been addressed in detailed; however, numerous mutations have been identified within SAUGI. Here, we investigated whether these mutations drastically perturb the interaction with SAUDG. To perform quantitative analysis of the macromolecular interactions, we applied native mass spectrometry and demonstrated that this is a highly efficient and specific method for determination of dissociation constants. Our results indicate that several naturally occurring mutations of SAUGI do indeed lead to appreciable changes in the dissociation constants for complex formation. However, all of these K d values remain in the nanomolar range and therefore the association of these two proteins is preserved. We conclude that complexation is most likely preserved even with the naturally occurring mutant uracil‐DNA glycosylase inhibitor proteins.

Lysophospholipid G protein-coupled receptor binding parameters as determined by backscattering interferometry

Lysophosphatidic acid (LPA) activates cognate G protein-coupled receptors (GPCRs) to initiate biological signaling cascades. Lysophospholipid (LP) receptor binding properties remain incompletely assessed because of difficulties with ligand lipophilicity and lipid "stickiness." These inherent attributes produce high levels of nonspecific binding within cell-membrane preparations used to assess GPCRs, as has been shown in classical binding assays using radiolabeled ligands, making accurate measurements of lipid binding kinetics difficult to achieve. Backscattering interferometry (BSI) is an optical technology that measures molecular binding interactions by reporting changes in the refractive index of a solution after binding events. Here, we report the use of BSI to assess LPA1 for its ability to bind to naturally occurring lipids and a synthetic LPA1 antagonist (ONO-9780307), under both primary- and competition-binding conditions. Assessment of 12 different lipids demonstrated that the known LP ligand, 1-oleoyl-LPA, as well as an endocannabinoid metabolite, anandamide phosphate, are specific ligands for LPA1, whereas other LPs tested were not. Newly determined dissociation constants (Kd values) for orthosteric lipid ligands approximated 10-9 M, substantially lower (i.e., with higher affinity) than measured Kd values in classical binding or cell-based assays. These results demonstrate that BSI may have particular utility in assessing binding interactions between lipid receptors and their lipid ligands and could provide new screening approaches for lipid receptor identification and drug discovery.

The polyanions heparin and suramin impede binding of free adenine to a DNA glycosylase from C. pseudotuberculosis

Currently no effective treatment is available to combat infections caused by Corynebacterium pseudotuberculosis in livestock. Survival of this Gram-positive bacterium in rapidly-growing pathogens in hostile environments is strongly dependent on the existence of a robust DNA repair system to prevent DNA mutations and contribute to bacterial colonization and virulence. The adenine/guanine-specific DNA glycosylase (MutY) is evolutionarily conserved and has been well characterized due to its central role in the prevention of mutagenesis and DNA repair.The aim of this study was the characterization of the target protein interaction with free adenine, suramin, and heparin, as well as the binding competition characterization between the molecules.The dissociation constant for free adenine interaction with Corynebacterium pseudotuberculosis MutY (Cp-MutY) was determined, 86 ± 2.5 μM. NMR competition experiments demonstrated, that the polyanions heparin and suramin compete with adenine for the protein active site. The determined dissociation constant for the heparin/Cp-MutY interaction was 5.9 ± 1.0 μM and for suramin was 16 ± 1.5 μM. Docking of both polyanions with Cp-MutY revealed a possible mode of interaction and indicates that these molecules can interfere with the protein interaction with damaged DNA or prevent the binding of the adenine base in the enzyme active site.

DNA Aptamers for the Malignant Transformation Marker CD24

Cluster of differentiation 24 (CD24) is a cell surface glycoprotein, which is largely present on hematopoietic cells and many types of solid tumor cells. CD24 is known to be involved in a wide range of downstream signaling pathways and neural development, yet the underlying mechanisms are poorly understood. Moreover, its production correlates with poor cancer prognosis, and targeting of CD24 with different antibodies has been shown to inhibit disease progression. Nucleic acid aptamers are oligonucleotides that are selected from random DNA or RNA libraries for high affinity and specific binding to a certain target. Thus, they can be used as an alternative to antibodies. To gain an insight on CD24 role and its interaction partners, we performed several SELEX (systematic evolution of ligands by exponential enrichment) experiments to select CD24-specfiic DNA aptamers. We found that the cell-SELEX approach was the most useful and that using HT-29 cell line presenting CD24 along with CD24 knockdown HT-29 cells has selected six aptamers. For the selected aptamers, we determined dissociation constants in the nanomolar range (18-709 nM) using flow cytometry. These aptamers can be applied as diagnostic tools to track cancer progression and bear a potential for therapeutic use for inhibiting signaling pathways that promote the metastatic process.

Stability constants for Zirconium(IV) complexes with EDTA, CDTA, and DTPA in perchloric acid solutions

We quantified the stability constants of Zr(IV) with ethylenediamine-N,N,N’,N’-tetraacetic acid (EDTA), trans-1,2-diaminocyclohexane-N,N,N’,N’-tetraacetic acid (CDTA), and diethylenetriamine-N,N,N’,N’’,N’’-pentaacetic acid (DTPA) in 0.75–1.00 mol∙L−1 HClO4 with 1.00 mol∙L−1 total ionic strength using a liquid-liquid extraction technique. The data indicated the formation of ubiquitous 1:1 complexes, but also newly reported 1:2 metal-ligand complexes. The 1:1 complexes were identified as Zr(EDTA)0 (log10β101 = 27.9 ± 0.1), Zr(CDTA)0 (log10β101 = 29.6 ± 0.2), and Zr(DTPA)− (log10β101 = 35.3 ± 0.3), and the newly identified bis-complexes as Zr(EDTA)24− (log10β102 = 54.4 ± 0.2), ZrH4(CDTA)20 (log10β142 = 58.5 ± 0.5), and ZrH8(DTPA)22+ (log10β182 = 70.3 ± 0.4). The quantification of the stability constants described above required determining acid dissociation constants for EDTA and DTPA at varied ionic strength at 25.0 ± 0.1 °C by potentiometric titration, and the Specific ion Interaction Theory (SIT) model was used to correlate the acid dissociation constants with ionic strength. The resulting thermodynamic constants at zero ionic strength from this analysis for EDTA were: pKa3° = 2.21 ± 0.06, pKa4° = 3.21 ± 0.06, pKa5° = 6.76 ± 0.09, and pKa6° = 10.27 ± 0.03, and for DTPA were: pKa4° = 2.31 ± 0.02, pKa5° = 3.23 ± 0.07, pKa6° = 4.96 ± 0.05, pKa7° = 9.51 ± 0.05, and pKa8° = 11.21 ± 0.05.

STD NMR as a Technique for Ligand Screening and Structural Studies.

STD NMR is a powerful ligand-based tool for screening small molecules and low molecular weight fragments for their interaction with a given macromolecule. Such information is invaluable both in the drug discovery sector and in understanding fundamental biological interactions. Recently, powerful methods have been developed to extract a greater wealth of information from the STD NMR experiment, including ligand binding epitopes, dissociation constant determination, and mapping of binding site properties. Herein we describe these STD NMR experiments, giving practical examples for each approach, and highlight the important parameters and common pitfalls that must be considered for a successful experiment.

Novel 1,4-naphthoquinone N-aroylthioureas: Syntheses, crystal structure, anion recognition properties, DFT studies and determination of acid dissociation constants

In this paper, five novel 1,4-naphthoquinone N-aroylthiourea derivatives were synthesized, by the reaction of 2,3-diaminonaphthalene-1,4-dione and aroylisothiocyanates, in excellent yield (88–96%) and characterized by using FT-IR, 1H/13C NMR and HRMS. In addition, the molecular structure of one of the 1,4-naphthoquinone N-aroylthiourea compounds was characterized by single crystal X-ray diffraction studies. The absorption spectra of the compounds were determined in DMSO and the dyes have absorption in visible region in the range of 464–477 nm. The chemosensing ability of each of the prepared dyes was assessed by titrating them towards the anions; F−, Cl−, Br−, I−, AcO−, CN−, ClO4−, H2PO4−, HSO4−, and NO3−, using spectrophotometric and 1H NMR titration techniques in DMSO, experimentally. The results obtained from the experimental investigation were explained by employing theoretical calculations. The molecular structure and absorption spectra of the compounds and their deprotonation forms were obtained using Density Functional Theory (DFT) and time-dependent DFT (TD-DFT) at B3LYP/6311 g(d) level of theory. The computed results are in good agreement with the experimental. Finally, the acid dissociation constants of the 1,4-naphthoquinone N-aroylthioureas were determined potentiometrically in 50% (v/v) DMSO-H2O hydro-organic solution at 25 ± 0.1 °C, at an ionic background of 0.1 mol/L of NaCl using the HYPERQUAD computer program.

Structural and thermal studies on some morpholine complexes

A series of mono metallic and hetero-metallic morpholine complexes were synthesized and characterized by elemental analysis, spectral (IR, UV-VIS and ESR) studies, room-temperature magnetic susceptibility and thermal analysis techniques(TG, DTG, DTA, DSC). IR spectra showed, that morpholine behaves as a monodentate ligand in all the complexes through nitrogen atom only except in case of [CuZn(MOR)3Cl4]·2H2O and [CoZn(MOR)5(H2O)2Cl4]·H2O complexes morpholine acts as a bidentate ligand through nitrogen and oxygen atoms. ESR was studied for copper complexes, were of isotropic nature. The geometry suggested to be octahedral in all complexes except in [CuZn(MOR)3Cl4]·2H2O and [CuNi(MOR)2Cl4]·4H2O complexes were tetrahedral. Molecular modeling techniques and quantum chemical methods have been done for morpholine to calculate charges, bond lengths, bond angles, dihedral angles, electronegativity (χ), chemical potential (μ), global hardness (η), softness (σ), and the electrophilicity index (ω). The thermal decomposition of the complexes and the ligand were monitored by TGA, DTG, DTA and DSC techniques under N2 atmosphere. The decomposition mechanisms were suggested. Studying the effect of pH on the electronic absorption spectra of morpholine, combined with determination of dissociation constants of morpholine (pKa1 = 5.283 ± 0.01, pKa2 = 8.373 ± 0.01). The biological activities of morpholine and all the complexes are tested against Gram positive and Gram negative bacteria. The results showed a well considerable activity for some complexes against different organisms.

Synthesis, antimycobacterial activity, and acid dissociation constants of polyfunctionalized 3-[2-(pyrrolidin-1-yl)thiazole-5-carbonyl]-2H-chromen-2-one derivatives

This study reports on synthesis and determination of antimycobacterial activity and acid dissociation constants of polyfunctionalized 3-[2-(pyrrolidin-1-yl)thiazole-5-carbonyl]-2H-chromen-2-one derivatives, containing thiazole, coumarin, and pyrrolidine octahydropyrrolo[3,4-c]pyrrole moieties. The products were synthesized by a cyclization reaction of 5,5-diphenylpyrrolidine N-aroylthioureas or methyl 5-substituted 4,6-dioxo-3,3-diphenyloctahydropyrrolo[3,4-c]pyrrole-1-carboxylate N-aroylthioureas and 3-(bromoacetyl)coumarin with good to excellent yield (81–97%). The compounds exhibited antimycobacterial activity against the M. tuberculosis H37Rv strain with minimum inhibitory concentration values in the range of 31.25–125 μg/cm3. Acid dissociation constants of the compounds were determined using data which were obtained using a potentiometric titration method in 50% (v/v) dimethyl sulfoxide–water hydroorganic solvent at 25 ± 0.1 °C, at an ionic background of 0.1 mol/dm3 of NaCl. Acid dissociation constants were calculated using the HYPERQUAD computer program. The acid dissociation constants obtained might be associated with SH, OH, and two NH groups, which were formed by the protonation of thiazole and pyrrolidine rings.

Multiwavelength UV-metric and pH-metric determination of the multiple dissociation constants of the lesinurad

Potentiometric and spectrophotometric pH-titrations of the lesinurad for three consecutive dissociation constants determination were compared. Lesinurad is a selective inhibitor of uric acid reabsorption as part of a combination of medicines to treat high levels of uric acid in blood, also called hyperuricemia. Nonlinear regression of the pH-spectra with REACTLAB and SQUAD84 and of the pH-titration curve with ESAB determined three multiple close dissociation constants. The protonation scheme of lesinurad was suggested. A sparingly soluble anion L− of lesinurad was protonated to the still soluble species LH, LH2+ and LH32+ in pure water. Three consecutive thermodynamic dissociation constants were estimated pKTa1 = 2.09, pKTa2 = 4.25, pKTa3 = 6.58 at 25 °C and pKTa1 = 1.96, pKTa2 = 4.16, pKTa3 = 6.32 at 37 °C by UV-metric spectra analysis. The graph of molar absorption coefficients shows that the spectrum of species LH2+ and LH vary in colour, while protonation of chromophore LH2+ to LH32+ has less influence on chromophores in the lesinurad molecule. Three multiple thermodynamic dissociation constants of 1 × 10−4 M lesinurad were determined by the pH-metric analysis pKTa1 = 2.39, pKTa2 = 3.47, pKTa3 = 6.17 at 25 °C and pKTa1 = 2.08, pKTa2 = 3.29, pKTa3 = 6.03 at 37 °C. The values of enthalpy ΔH0(pKa1) = 19.19 kJ mol−1, ΔH0(pKa2) = 13.29 kJ mol−1, ΔH0(pKa3) = 38.39 kJ mol−1, show the dissociation process is endothermic. The positive values of ΔG0(pKa1) = 11.93 kJ mol−1, ΔG0(pKa2) = 24.26 kJ mol−1, ΔG0(pKa3) = 37.56 kJ mol−1 at 25 °C indicate that the dissociation process of pKa2 is not spontaneous, which was confirmed by its value of entropy ΔS0(pKa1) = 24.37 J mol−1, ΔS0(pKa2) = −36.79 J mol−1, ΔS0(pKa3) = 2.79 J mol−1. Three macro-dissociation constants of lesinurad and protonation locations were predicted by MARVIN and ACD/Percepta.

Multiple dissociation constants of the Intepirdine hydrochloride using regression of multiwavelength spectrophotometric pH-titration data

Spectrophotometric and potentiometric pH-titrations of the Neurotransmitter Intepirdine hydrochloride INN·HCl for three dissociation constants determination were compared. A nonlinear regression of the pH-spectra (REACTLAB, SQUAD84) and of the pH-titration curve (ESAB) determined three multiple dissociation constants. A sparingly soluble neutral molecule LH of INN·HCl was capable of protonation to form the still soluble three cations LH2+, LH32+ and LH43+ in pure water. Although the change of pH somewhat less affected changes in the chromophore, three consecutive thermodynamic dissociation constants were estimated pKTa1=5.64, pKTa2=7.31, pKTa3=8.85 at 25°C and pKTa1=5.51, pKTa2=7.15, pKTa3=8.77 at 37°C. The graph of molar absorption coefficients of variously protonated species according to wavelength shows that the spectrum of species LH2+ and LH vary in colour, while protonation of chromophore LH2+ to LH32+ and LH43+ has less influence on chromophores in Intepirdine hydrochloride molecule. As the spectral response on the chromophore in the INN·HCl molecule is not large, it was necessary to test a reliability whether it is possible to estimate the dissociation constants even from such small spectrum changes. Three multiple thermodynamic dissociation constants of 3×10−4M INN·HCl were determined by the regression analysis of potentiometric titration curves pKTa1=5.14, pKTa2=8.38, pKTa3=9.33 at 25°C and pKTa1=5.17, pKTa2=8.31, pKTa3=9.07 at 37°C. The macro-dissociation constants of INN·HCl were estimated according to the chemical structure analyzed by two pKa predictors, the MARVIN and ACD/Percepta programs. The resulting protonation scheme of INN·HCl was suggested.

Effect of Mg and Ca on the Stability of the MRI Contrast Agent Gd–DTPA in Seawater

Gadolinium diethylenetriaminepentaacetic acid (Gd–DTPA) is widely applied as a contrast enhancer in medical MRI. As Gd–DTPA is only minimally captured in wastewater treatment plants (WTPs) or degraded by UV light and other oxidative processes, concentrations in rivers have increased globally by orders of magnitude following its introduction in 1987. The complex also seems impervious to estuarine scavenging and is beginning to emerge in coastal waters, yet it is unknown how its stability is changed by competition for the DTPA ligand from major seawater cations. We performed potentiometric titrations at seawater ionic strength (0.7 M NaClO4) to determine dissociation constants of the five DTPA carboxylic acid groups, as well as stability constants of Mg, Ca, and Gd complexes with the fully deprotonated and single-protonated ligand. These are in general agreement with literature values at low ionic strength and confirm that complexes with Ca are more stable than with Mg. A new finding, that the DTPA complexes of Mg and Ca appear to be hydrolyzed at elevated pH, implies that their coordination in these chelates is less than hexadentate, enabling additional competition with Gd from dinuclear Mg and Ca species. Side-reaction coefficients for trace-metal-free seawater, calculated from our results, suggest that the higher abundance of Mg and Ca may significantly destabilize Gd–DTPA in coastal waters, causing dissociation and release of as much as 15% of the organically complexed Gd from the ligand. This effect could enhance the particle-reactivity and bioavailability of anthropogenic Gd in sensitive estuarine habitats, indicating an urgent need to further study the fate of this contaminant in marine environments.

Pulse EPR distance measurements to study multimers and multimerisation

ABSTRACTPulse dipolar electron paramagnetic resonance (PD-EPR) has become a powerful tool for structural biology determining distances on the nanometre scale. Recent advances in hardware, methodology, and data analysis have widened the scope to complex biological systems. PD-EPR can be applied to systems containing lowly populated conformers or displaying large intrinsic flexibility, making them all but intractable for cryo-electron microscopy and crystallography. Membrane protein applications are of particular interest due to the intrinsic difficulties for obtaining high-resolution structures of all relevant conformations. Many drug targets involved in critical cell functions are multimeric channels or transporters. Here, common approaches for introducing spin labels for PD-EPR cause the presence of more than two electron spins per multimeric complex. This requires careful experimental design to overcome detrimental multi-spin effects and to secure sufficient distance resolution in presence of multiple distances. In addition to obtaining mere distances, PD-EPR can also provide information on multimerisation degrees allowing to study binding equilibria and to determine dissociation constants.

Determination of Dissociation Constant (PKA) of Enalapril maleate by Electromagnetic Instrument Based Method

A simple, rapid and precise Electromagnetic Instrument Based Method (Conductometry and Potentiometry) has been developed for the determination of Dissociation constant. Dissociation constant is playing a great role in the drug absorption in vivo, especially in case of oral administration. So, in drug designing, pKa i.e. the negative logarithm of dissociation constant determination is very important. In this regards we report electromagnetic types of instrument-based procedures with principles elaborated to determine the Pka of Enalapril Maleate, an ACE inhibitor type of antihypertensive drug. The Dissociation Constant (PKa) of Enalapril Maleate was found to be 2.7 and 2.6 by the Potentiometric titration and Conductometry methods respectively.

Spectrophotometric pH measurements in the presence of dissolved organic matter and hydrogen sulfide

AbstractSpectrophotometric pH measurements were first introduced for oceanic environments and they facilitated the determination of the marine CO2 system, including the direct observation of Ocean Acidification. Extended characterizations of the indicator dye m‐Cresol purple over the past two decades enabled the application of this method to natural waters ranging from brines to freshwaters. However, the required determination of the dye's dissociation constants and absorbance properties were exclusively performed in buffer solutions prepared with artificial seawater. Potential perturbations by substances that occur in natural waters, but are not included in the buffer solutions, have never been tested. Therefore, we studied the impact of elevated amounts of dissolved organic matter (DOM) and hydrogen sulfide (H2S) on spectrophotometric pH measurements. We did not observe an impact on spectrophotometric pH measurements by H2S concentrations up to 400 μmol kg−1, which reflect high levels such as those reported from the Black Sea. Likewise, natural DOM did not interfere with the spectrophotometric measurements at concentrations typical for oceanic environments and large estuarine systems. However, strongly colored river waters can cause spectral disturbances resulting in calculated pH values that are up to tenths of pH units too low. To circumvent such disturbances, we recommend using intense light sources, a shorter cuvette length or spectrophotometrically calibrated glass electrodes when performing spectrophotometric measurements under critical conditions.

40 - Redox modulation of hSIRT6, key enzyme of metabolism and inflammation

Sirtuins are NAD+-dependent deacetylases reported as key factors in metabolism regulation, inflammation and stress response. Human sirtuin 6 (hSIRT6) is known as a histone 3 deacetylase despite of its low activity in vitro. Its deacetylase activity increases in the presence of fatty acids and possess long chain fatty acid deacylase activity. Studies on regulation of hSIRT6 activity are scarce. Our aim was to investigate the potential redox regulation of hSIRT6, characterizing structural and functional changes under different redox conditions. Recombinant hSIRT6 was expressed and purified from E. coli. Deacylase activity was measured using a coupled-enzyme assay (plus nicotinamidase and glutamate dehydrogenase under non-limiting conditions) following NADPH consumption at 340 nm. Synthetic acetylated and myristoylated histone 3 peptides (H3K9-Ac, H3K9-Myr) were used as substrates. Total protein thiols were oxidized after hSIRT6 treatment with H2O2 (50x, 10 min), however, deacetylase activity was not significantly altered, while treatment with an excess of peroxynitrite reduced protein thiol content with a significant loss of activity. The increase in basal deacetylase activity in the presence of 100 μM oleic acid was confirmed. Surprisingly, treatment with the electrophilic derivative nitro-oleic acid do not inactivate, on the contrary, enhanced deacetylase activity, albeit the presence of a catalytic histidine. Other nitro-fatty acids gave the same response. Thermodynamic analysis with determination of dissociation constants is underway.

Study of in vitro transcriptional binding effects and noise using constitutive promoters combined with UP element sequences in Escherichia coli

BackgroundUP elements (upstream element) are DNA sequences upstream of a promoter that interact with the α-subunit of RNA polymerase (RNAP) and can affect transcription by altering the binding RNAP to DNA. However, details of UP element and binding affinity effects on transcriptional strength are unclear.ResultsHere, we investigated the effects of UP element sequences on gene transcription, binding affinity, and gene expression noise. Addition of UP elements resulted in increased gene expression (maximum 95.7-fold increase) and reduced gene expression noise (8.51-fold reduction). Half UP element sequences at the proximal subsite has little effect on transcriptional strength despite increasing binding affinity by 2.28-fold. In vitro binding assays were used to determine dissociation constants (Kd) and in the in vitro system, the full range of gene expression occurs in a small range of dissociation constants (25 nM < Kd < 45 nM) indicating that transcriptional strength is highly sensitive to small changes in binding affinity.ConclusionsThese results demonstrate the utility of UP elements and provide mechanistic insight into the functional relationship between binding affinity and transcription. Given the centrality of gene expression via transcription to biology, additional insight into transcriptional mechanisms can foster both fundamental and applied research. In particular, knowledge of the DNA sequence-specific effects on expression strength can aid in promoter engineering for different organisms and for metabolic engineering to balance pathway fluxes.

CYP109E1 is a novel versatile statin and terpene oxidase from Bacillus megaterium.

CYP109E1 is a cytochromeP450 monooxygenase from Bacillus megaterium with a hydroxylation activity for testosterone and vitamin D3. This study reports the screening of a focused library of statins, terpene-derived and steroidal compounds to explore the substrate spectrum of this enzyme. Catalytic activity of CYP109E1 towards the statin drug-precursor compactin and the prodrugs lovastatin and simvastatin as well as biotechnologically relevant terpene compounds including ionones, nootkatone, isolongifolen-9-one, damascones, and β-damascenone was found in vitro. The novel substrates induced a type I spin-shift upon binding to P450 and thus permitted to determine dissociation constants. For the identification of conversion products by NMR spectroscopy, a B. megaterium whole-cell system was applied. NMR analysis revealed for the first time the ability of CYP109E1 to catalyze an industrially highly important reaction, the production of pravastatin from compactin, as well as regioselective oxidations generating drug metabolites (6'β-hydroxy-lovastatin, 3'α-hydroxy-simvastatin, and 4″-hydroxy-simvastatin) and valuable terpene derivatives (3-hydroxy-α-ionone, 4-hydroxy-β-ionone, 11,12-epoxy-nootkatone, 4(R)-hydroxy-isolongifolen-9-one, 3-hydroxy-α-damascone, 4-hydroxy-β-damascone, and 3,4-epoxy-β-damascone). Besides that, a novel compound, 2-hydroxy-β-damascenone, produced by CYP109E1 was identified. Docking calculations using the crystal structure of CYP109E1 rationalized the experimentally observed regioselective hydroxylation and identified important amino acid residues for statin and terpene binding.

Affinity capillary electrophoresis for the assessment of binding affinity of carbohydrate-based cholera toxin inhibitors.

Developing tools for the study of protein carbohydrate interactions is an important goal in glycobiology. Cholera toxin inhibition is an interesting target in this context, as its inhibition may help to fight against cholera. For the study of novel ligands an affinity capillary electrophoresis (ACE) method was optimized and applied. The method uses unlabeled cholera toxin B-subunit (CTB) and unlabeled carbohydrate ligands based on ganglioside GM1-oligosaccharides (GM1os). In an optimized method at pH4, adsorption of the protein to the capillary walls was prevented by a polybrene-dextran sulfate-polybrene coating. Different concentrations of the ligands were added to the BGE. CTB binding was observed by a mobility shift that could be used for dissociation constant (Kd ) determination. The Kd values of two GM1 derivatives differed by close to an order of magnitude (600±20nM and 90±50nM) which was in good agreement with the differences in their reported nanomolar IC50 values of an ELISA-type assay. Moreover, the selectivity of GM1os towards CTB was demonstrated using Influenza hemagglutinin (H5) as a binding competitor. The developed method can be an important platform for preclinical development of drugs targeting pathogen-induced secretory diarrhea.