Optimization of reorganization energy drives evolution of the designed Kemp eliminase KE07.

Understanding enzymatic evolution is essential to engineer enzymes with improved activities or to generate enzymes with tailor-made activities. The computationally designed Kemp eliminase KE07 carries out an unnatural reaction by converting of 5-nitrobenzisoxazole to cyanophenol, but its catalytic efficiency is significantly lower than those of natural enzymes. Three series of designed Kemp eliminases (KE07, KE70, KE59) were shown to be evolvable with considerable improvement in catalytic efficiency. Here we use the KE07 enzyme as a model system to reveal those forces, which govern enzymatic evolution and elucidate the key factors for improving activity. We applied the Empirical Valence Bond (EVB) method to construct the free energy pathway of the reaction in the original KE07 design and the evolved R7 1/3H variant. We analyzed catalytic effect of residues and demonstrated that not all mutations in evolution are favorable for activity. In contrast to the small decrease in the activation barrier, in vitro evolution significantly reduced the reorganization energy. We developed an algorithm to evaluate group contributions to the reorganization energy and used this approach to screen for KE07 variants with potential for improvement. We aimed to identify those mutations that facilitate enzymatic evolution, but might not directly increase catalytic efficiency. Computational results in accord with experimental data show that all mutations, which appear during in vitro evolution were either neutral or favorable for the reorganization energy. These results underscore that distant mutations can also play role in optimizing efficiency via their contribution to the reorganization energy. Exploiting this principle could be a promising strategy for computer-aided enzyme design. This article is part of a Special Issue entitled: The emerging dynamic view of proteins: Protein plasticity in allostery, evolution and self-assembly.

[Alveolar microlithiasis: an uncommon cause of the crazy paving pattern]. / Microlitiasis alveolar: presentación infrecuente del "patrón en empedrado"

Alveolar microlithiasis is an uncommon disease of unknown etiology characterized by the presence of multiple, predominantly subpleural, intra-alveolar microcalcifications. We present a case in which the high-resolution CT images show diffuse pulmonary microcalcifications together with patchy areas of ground glass attenuation associated to marked thickening of the interlobular septa, all of which taken together constitute the crazy paving pattern. This pattern is not specific for alveolar microlithiasis; it has also been reported in other entities, including alveolar proteinosis, lipoid pneumonia, and bronchial alveolar carcinoma.

Torsion of a wandering accessory spleen: CT findings.

Torsion of an accessory spleen is a rare entity that can have a variable clinical presentation. We report the computed tomographic (CT) findings of an acute torsion of an accessory spleen in a 13-year-old girl. CT disclosed a hypodense mesenteric mass with peripheral inflammatory changes.