Kemp Eliminases of the AlleyCat Family Possess High Substrate Promiscuity.

Minimalist enzymes designed to catalyze model reactions provide useful starting points for creating catalysts for practically important chemical transformations. We have shown that Kemp eliminases of the AlleyCat family facilitate conversion of leflunomide (an immunosupressor pro-drug) to its active form teriflunomide with outstanding rate enhancement (nearly four orders of magnitude) and catalytic proficiency (more than seven orders of magnitude) without any additional optimization. This remarkable activity is achieved by properly positioning the substrate in close proximity to the catalytic glutamate with very high pKa.

Catalytic Nanoassemblies Formed by Short Peptides Promote Highly Enantioselective Transfer Hydrogenation.

Self-assembly enables formation of incredibly diverse supramolecular structures with practically important functions from simple and inexpensive building blocks. Here, we show how a semirational, bottom-up approach to create emerging properties can be extended to a design of highly enantioselective catalytic nanoassemblies. The designed peptides comprising as few as two amino acid residues spontaneously self-assemble in the presence of metal ions to form supramolecular, vesicle-like nanoassemblies that promote transfer hydrogenation of ketones in an aqueous phase with excellent conversion rates and enantioselectivities (>90% ee).

Copper-Containing Catalytic Amyloids Promote Phosphoester Hydrolysis and Tandem Reactions.

Self-assembly of short de novo designed peptides gives rise to catalytic amyloids capable of facilitating multiple chemical transformations. We show that catalytic amyloids can efficiently hydrolyze paraoxon, a widely used, highly toxic organophosphate pesticide. Moreover, these robust and inexpensive metal-containing materials can be easily deposited on various surfaces producing catalytic flow devices. Finally, functional promiscuity of catalytic amyloids promotes tandem hydrolysis/oxidation reactions. High efficiency discovered in a very small library of peptides suggests an enormous potential for further improvement of catalytic properties both in terms of catalytic efficiency and substrate scope.

The effect of conjugation on antitumor activity of vindoline derivatives with octaarginine, a cell-penetrating peptide.

Some Vinca alkaloids (eg, vinblastine, vincristine) have been widely used as antitumor drugs for a long time. Unfortunately, vindoline, a main alkaloid component of Catharanthus roseus (L.) G. Don, itself, has no antitumor activity. In our novel research program, we have prepared and identified new vindoline derivatives with moderate cytostatic activity. Here, we describe the effect of conjugation of vindoline derivative with oligoarginine (tetra-, hexa-, or octapeptides) cell-penetrating peptides on the cytostatic activity in vitro and in vivo. Br-Vindoline-(l)-Trp-OH attached to the N-terminus of octaarginine was the most effective compound in vitro on HL-60 cell line. Analysis of the in vitro activity of two isomer conjugates (Br-vindoline-(l)-Trp-Arg8 and Br-vindoline-(d)-Trp-Arg8 suggests the covalent attachment of the vindoline derivatives to octaarginine increased the antitumor activity significantly against P388 and C26 tumour cells in vitro. The cytostatic effect was dependent on the presence and configuration of Trp in the conjugate as well as on the cell line studied. The configuration of Trp notably influenced the activity on C26 and P388 cells: conjugate with (l)-Trp was more active than conjugate with the (d)-isomer. In contrast, conjugates had very similar effect on both the HL-60 and MDA-MB-231 cells. In preliminary experiments, conjugate Br-vindoline-(l)-Trp-Arg8 exhibited some inhibitory effect on the tumor growth in P388 mouse leukemia tumor-bearing mice. Our results indicate that the conjugation of modified vindoline could result in an effective compound even with in vivo antitumor activity.

Preparation and Screening of Catalytic Amyloid Assemblies.

Aggregation of proteins into amyloids has long been recognized as one of the major contributors to disease and aging. Amyloids are known to catalyze their own formation but they have been considered the rock-bottom thermodynamic minimum of the protein fold without much functionality. We have recently demonstrated that aggregation of short peptides in the presence of metal ions gives rise to efficient catalytic activity. Here we present a detailed protocol for the synthesis and purification of these peptides and the preparation of amyloid-like fibrils. Then we describe an easy-to-perform, high-throughput assay to measure their hydrolytic activity.

Functional tuning of the catalytic residue pKa in a de novo designed esterase.

AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p-nitrophenyl acetate (pNPA) and 4-nitrophenyl-(2-phenyl)-propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single-histidine residue (His144 ) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pKa value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pKa calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pKa value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pKa value of this residue in water. We determined that a short-range favorable interaction with Glu127 contributes to the elevated pKa of His144 . We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pKa of its active nucleophile, His144 , by 0.7 units. As a direct result of the decrease in the His144 pKa value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine-promoted catalysis. Proteins 2017; 85:1656-1665. © 2017 Wiley Periodicals, Inc.

Acetochlor as a soil pollutant.

Acetochlor is a widely used herbicide all over the world. Similarly to other organic pollutants, the environmental fate of the acetochlor is strongly related to its adsorption properties. Static adsorption equilibrium measurements were carried out at 25 degrees C on different types of Hungarian soils characterized by varying amounts of organic matter and pH values. Isotherms obtained under different conditions, as well as on various soils, exhibit a similar shape, thus indicating a two-step adsorption process. The plots cannot be interpreted according to the classes of isotherms suggested by Giles. The adsorption coefficients (K) were estimated from the initial slope of the curves. These values were determined not only by the type of the soil, but also by the composition of the aqueous media. The organic matter adsorption coefficients (Kom) were also calculated and they were approximately identical for soils of high organic matter. Due to the low value of the adsorption coefficients, the acetochlor is a rather mobile pollutant of the soil posing a potential danger to the aquatic environment. For soils with high organic content, the Kom values are similar to each other and, due to the significant coverage of the inorganic surfaces, the adsorption is controlled by the solute-organic substance interactions. The adsorption of weakly polar compounds, however, is remarkable even on those soils where the organic content is low. In this case, the binding is governed by the solute-inorganic surface interactions. This conclusion is nicely proved by the adsorption of the acetochlor on quartz. According to our hypothesis, the second part of the adsorption step is controlled by the solute-adsorbed solute interactions. The role of the organic matter in this region of the isotherm is probably negligible. As the organic matter content of the soils plays an important role in the acetochlor adsorption, humic substances must have a strong influence upon the transport of this compound. Experiments to obtain adsorption isotherms of further pesticides and the development of a quantitative model are in progress.

Adsorption of propisochlor on soils and soil components equation for multi-step isotherms.

Static equilibrium measurements were performed for the study of propisochlor on three different kinds of soils at pH = 7.0 at 25 degrees C. The concentration of herbicide was changed from 30 to 300 mumol/l. The obtained two-step adsorption isotherms cannot be evaluated by using the Freundlich or Langmuir equations. New equation has been derived by making use of the usual mass balance and equilibrium relationships of the adsorption and by considering the possibility of the formation of associates of the hydrophobic solute molecules. The characteristic model parameters of each step of the adsorption isotherm were estimated for the studied systems by a non-linear least square regression. The calculated curves fit well to the experimentally obtained two-step isotherms and the parameters of the model can be used for the characterization of the pesticide-soil interactions and consequently the mobility of the propisochlor in soil/water systems.