High resolution x-ray crystallographic structures of nitrite reductase from Achromobacter cycloclastes, undertaken in order to understand the pH optimum of the reaction with nitrite, show that at pH 5.0, 5.4, 6.0, 6.2, and 6.8, no significant changes occur, other than in the occupancy of the type II copper at the active site. An extensive network of hydrogen bonds, both within and between subunits of the trimer, maintains the rigidity of the protein structure. A water occupies a site approximately 1.5 A from the site of the type II copper in the structure of the type II copper-depleted structure (at pH 5.4), again with no other significant changes in structure. In nitrite-soaked crystals, nitrite binds via its oxygens to the type II copper and replaces the water normally bound to the type II copper. The active-site cavity of the protein is distinctly hydrophobic on one side and hydrophilic on the other, providing a possible path for diffusion of the product NO. Asp-98 exhibits thermal parameter values higher than its surroundings, suggesting a role in shuttling the two protons necessary for the overall reaction. The strong structural homology with cupredoxins is described.
The three-dimensional crystal structure of the copper-containing nitrite reductase (NIR) from Achromobacter cycloclastes has been determined to 2.3 angstrom (A) resolution by isomorphous replacement. The monomer has two Greek key beta-barrel domains similar to that of plastocyanin and contains two copper sites. The enzyme is a trimer both in the crystal and in solution. The two copper atoms in the monomer comprise one type I copper site (Cu-I; two His, one Cys, and one Met ligands) and one putative type II copper site (Cu-II; three His and one solvent ligands). Although ligated by adjacent amino acids Cu-I and Cu-II are approximately 12.5 A apart. Cu-II is bound with nearly perfect tetrahedral geometry by residues not within a single monomer, but from each of two monomers of the trimer. The Cu-II site is at the bottom of a 12 A deep solvent channel and is the site to which the substrate (NO2-) binds, as evidenced by difference density maps of substrate-soaked and native crystals.
A method is introduced to calculate and compare the variability of molecular descriptors in compound databases. Descriptor variability analysis is based on histograms recording the distribution of molecular descriptors and calculation of Shannon entropy (SE), a metric originally applied in digital communication. SE values reflect the variability of descriptor settings. We have calculated a total of 92 molecular descriptors in the ACD and NCI databases and ranked them according to their variability. Significant differences in entropy are observed for a number of descriptors. However, the most variable descriptors are similar in the ACD and NCI databases. Such high-entropy descriptors are preferred tools to discriminate between compounds or account for the diversity of chemical libraries.
A combinatorial method was developed to calculate complete distributions of the Tanimoto coefficient (Tc) for binary fingerprint (FP) representations of specified length, regardless of the chemical parameters they reflect. Theoretical Tc distributions were calculated for FPs consisting of up to 67 bit positions which revealed significant statistical preferences of certain Tc values. Calculation of Tc distributions in a large compound database using different FPs mirrored the effects identified by our general analysis. On the basis of these findings, an average Tc is biased by statistically preferred values.
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