Dr Susan Crennell
Structures of molecules from organisms adapted to extreme environments
Citrate synthase has been the model molecule used by the CER to identify structural features related to thermostability, and the X-ray structures of citrate synthase with temperature optima ranging from 5oC to 100oC were determined over a number of years by previous members of the CER. These had been compared to the mesophilic pig and chicken CS structures and a number of features identified as possibly contributing to thermostability. To provide a mesophilic comparator from a microorganism, the crystal structure of Bacillus subtilis was determined. (Christian Vogl). This is indeed more similar to the other micro-organismal CS than those from pig or chicken. We are mining the family of structures using a number of computational techniques to identify features which may be responsible for the thermostability. Hypotheses arising from computer-based studies will be tested through site-directed mutagenesis.
The crystal structure of Bacillus subtilis CS. It is a dimer, each monomer consisting of two domains, a large domain (blue and yellow) which forms the majority of the dimer interface, and a small domain (pink and green) which move to close round the substrate during catalysis.
Other enzymes of interest are those produced by the thermophile Rhodothermus marinus for plant cell well degradation (collaboration with Eva Nordberg-Karlsson of Lund University). Multi-domain Xylanase Xyn10A has been the subject of protein-protein interaction predictions and small angle X-ray scattering (SAXS) experiments to reveal the relative location of the domains in the structure. The X-ray crystal structure of hyperthermophilic cellulase Cel12A when compared to mesophilic homologues revealed a number of features which may result in the very high thermal stability (half life of more than 2 hours at 90oC). Structures of the enzyme in complex with substrate have also been obtained and are being analysed to shed light on the high temperature optimum (also > 90oC).
Two perpendicular views of the results of SAXS analysis of Xyn10A. Averaged low resolution models shown as blue beads, typical DR models displayed as green spheres and the models obtained by rigid body refinement represented as red Calpha-traces. (Analysis by D. Svergun, EMBL Hamburg)
Structure of Cel12A with substrate (cellotetraose, drawn with yellow bonds) bound in the active site.