An alternate sucrose binding mode in the E203Q Arabidopsis invertase mutant: an X-ray crystallography and docking study

In the present study, we report on the X-ray crystallographic structure of a GH32 invertase mutant, (i.e., the Arabidopsis thaliana cell-wall invertase 1-E203Q, AtcwINV1-mutant) in complex with sucrose. This structure was solved to reveal the features of sugar binding in the catalytic pocket. However, as demonstrated by the X-ray structure the sugar binding and the catalytic pocket arrangement is significantly altered as compared with what was expected based on previous X-ray structures on GH-J clan enzymes. We performed a series of docking and molecular dynamics simulations on various derivatives of AtcwINV1 to reveal the reasons behind this modified sugar binding. Our results demonstrate that the E203Q mutation introduced into the catalytic pocket triggers conformational changes that alter the wild type substrate binding. In addition, this study also reveals the putative productive sucrose binding modus in the wild type enzyme.     

P2-P1' macrocyclization of P2 phenylglycine based HCV NS3 protease inhibitors using ring-closing metathesis

Macrocyclization is a commonly used strategy to preorganize HCV NS3 protease inhibitors in their bioactive conformation. Moreover, macrocyclization generally leads to greater stability and improved pharmacokinetic properties. In HCV NS3 protease inhibitors, it has been shown to be beneficial to include a vinylated phenylglycine in the P2 position in combination with alkenylic P1' substituents. A series of 14-, 15- and 16-membered macrocyclic HCV NS3 protease inhibitors with the linker connecting the P2 phenylglycine and the alkenylic P1' were synthesized by ring-closing metathesis, using both microwave and conventional heating. Besides formation of the expected macrocycles in cis and trans configuration as major products, both ring-contracted and double-bond migrated isomers were obtained, in particular during formation of the smaller rings (14- and 15-membered rings). All inhibitors had K(i)-values in the nanomolar range, but only one inhibitor type was improved by rigidification. The loss in inhibitory effect can be attributed to a disruption of the beneficial π-π interaction between the P2 fragment and H57, which proved to be especially deleterious for the d-phenylglycine epimers.     

Inferring the sources of postglacial range expansion in two large European land snails

Exact locations of glacial refugia are relevant for the study of contemporary biodiversity, not only as places less disturbed during the climatic changes but also as sources of rapid expansion of the biota after the Last Glacial cycle. If continuously inhabited over several of the Quaternary glacial cycles, the refugia are readily identifiable by the accumulated genetic diversity. However, the sources of the Holocene range expansion, particularly important for the emergence of present-day bio- and phylogeographic patterns and for realistic estimation of species’ expansion rates, might have been located at the fringes of the glacial species ranges and lack unique lineages. This problem is pertinent when the variation is explored at slowly evolving genetic markers. We suggest that the location of such source refugia may be approximated by reconstructing the geographic location as a continuous trait evolving along the branches of a phylogenetic tree. We applied this approach, using the BEAST software, on two large southeast European land snail species: Caucasotachea vindobonensis and Helix thessalica. We found evidence for C. vindobonensis refugia in the western Balkans; notable is an apparently old refugium in Bosnia and Herzegovina. The plausible sources of the species’ Holocene range expansion, however, were located around the south-western end of the Carpathians. Although the source areas were likely similar in H. thessalica, some expansion sources suggested by the analyses (e.g., Podolia, Ukraine) appeared implausible and driven by sampling clustered in that area. The applied approach allows for additional exploitation of the mitochondrial data gathered during the past two decades of animal phylogeography studies.