A challenge in structural genomics is prediction of the function of uncharacterized proteins. When proteins cannot be related to other proteins of known activity, identification of function based on sequence or structural homology is impossible and in such cases it would be useful to assess structurally conserved binding sites in connection with the protein's function. In this paper, we propose the function of a protein of unknown activity, the Tm1631 protein from Thermotoga maritima, by comparing its predicted binding site to a library containing thousands of candidate structures. The comparison revealed numerous similarities with nucleotide binding sites including specifically, a DNA-binding site of endonuclease IV. We constructed a model of this Tm1631 protein with a DNA-ligand from the newly found similar binding site using ProBiS, and validated this model by molecular dynamics. The interactions predicted by the Tm1631-DNA model corresponded to those known to be important in endonuclease IV-DNA complex model and the corresponding binding free energies, calculated from these models were in close agreement. We thus propose that Tm1631 is a DNA binding enzyme with endonuclease activity that recognizes DNA lesions in which at least two consecutive nucleotides are unpaired. Our approach is general, and can be applied to any protein of unknown function. It might also be useful to guide experimental determination of function of uncharacterized proteins.

Elevated expression of the immunoproteasome has been associated with autoimmune diseases, inflammatory diseases, and various types of cancer. Selective inhibitors of the immunoproteasome are not only scarce, but also almost entirely restricted to peptide-based compounds. Herein, we describe nonpeptidic reversible inhibitors that selectively block the chymotrypsin-like (β5i) subunit of the human immunoproteasome in the low micromolar range. The most potent of the reversibly acting compounds were then converted into covalent, irreversible, nonpeptidic inhibitors that retained selectivity for the β5i subunit. In addition, these inhibitors discriminate between the immunoproteasome and the constitutive proteasome in cell-based assays. Along with their lack of cytotoxicity, these data point to these nonpeptidic compounds being suitable for further investigation as β5i-selective probes for possible application in noncancer diseases related to the immunoproteasome.

Autolysin E (AtlE), from Staphylococcus aureus, is a cell-wall-degrading enzyme that is a potential drug target. It is a member of the glycoside hydrolase (GH) class, enzymes that commonly have either two catalytic residues and hydrolyze their substrates by inverting or retaining mechanisms or one catalytic residue and undergo retaining, substrate-assisted catalysis. Here, we address the catalytic mechanism of AtlE. Site-directed mutagenesis studies identified Glu138 as the only catalytic residue. Quantum mechanics/molecular mechanics (QM/MM) simulations of the possible reaction pathways suggest that hydrolysis proceeds via a retaining, water-assisted mechanism and an oxocarbenium ion like transition state. These results, on the basis of data from a member of the hydrolase GH73 family, support the hypothesis of the presence of an alternative catalytic mechanism in glycoside hydrolases, which can be considered in the design of future AtlE inhibitors.

An optimal multiscale linking and integration of atom-scale density functional theory (DFT) computations with mesoscopic kinetic Monte Carlo (KMC) is gaining in importance, particularly upon considering the engineering and intensification of unconventional feedstock processing, as well as the design of emerging catalysis routes. Carbon dioxide activation for methanol synthesis reactions on Cu(111) catalysts was studied using first-principles calculations and KMC modeling simulations. The CO2 hydrogenation pathway model was applied, consisting of the formate and the reverse water–gas shift (RWGS) mechanistic steps. The dependence of conversion, selectivity, and the rate of desorbed bulk CH3OH production upon operating process conditions, primarily temperature and pressure, was examined. Catalytic performance results are qualitatively well comparable with the available literature data for heterogeneous copper-based materials. Furthermore, the numerical stability analysis of KMC simulations was statistically assessed with respect to random seed parameters and activation energy barriers. Surface product distribution was found to be particularly sensitive to the smallest perturbations of the activation standard Gibbs energy. The effects of binding site size, crystal lattice dimensions, packed-bed influx composition (gaseous phase reactant partial pressures), and input randomized numbers were, however, less pronounced. This demonstrates that an accurate evaluation of ab initio theoretical research is crucial, especially upon paralleling them to experimental reactor concentrations.

Elevated expression of the immunoproteasome has been associated with autoimmune diseases,i nflammatory diseases,a nd various types of cancer.S elective inhibitors of the immunoproteasome are not only scarce,b ut also almost entirely restricted to peptide-based compounds.H erein, we describe nonpeptidic reversible inhibitors that selectively block the chymotrypsin-like (b5i)s ubunit of the human immunoproteasome in the low micromolar range.T he most potent of the reversibly acting compounds were then converted into covalent, irreversible,n onpeptidic inhibitors that retained selectivity for the b5i subunit. In addition, these inhibitors discriminate between the immunoproteasome and the constitutive proteasome in cell-based assays.Along with their lack of cytotoxicity,t hese data point to these nonpeptidic compounds being suitable for further investigation as b5i-selective probes for possible application in noncancer diseases related to the immunoproteasome.