| Natural evolution gave rise to all currently living species by continuous adaptation of the organisms and of their molecular constituents to their environment. This process is studied in laboratory using directed evolution at the cellular or molecular level and judicious experimental design. In the field of accelerated evolution of proteins, the potential of oligomeric proteins has been largely under-exploited despite their importance in nature. Up to now, little information is available about gene duplication, mutations accumulation in both gene copies and association of their products into homo- and hetero-oligomers. Moreover, the modular nature of oligomeric proteins allows the construction of large diversity libraries by combination of smaller ones. This fact increases the explored mutational landscape and the chances to find interesting mutants. Since an important challenge in protein engineering is the directed evolution of enzymes, we chose three homologous isocitrate dehydrogenases (IDH) with different thermodynamic properties as potential dimeric evolution platforms: the mesophilic Escherichia coli IDH, the thermostable IDH from Archaeoglobus fulgidus and the flexible but stable IDH from the psychrophile Desulfotalea psychrophila. We work in engineering the structure and the activity of these model proteins. On the one hand, we showed that subunits issued from different organisms may assemble into functional heterodimers. We’re further trying to characterize and improve this interaction by directed evolution. On the other hand, enzymes with modified substrate specificity to L-malate will be selected thanks to an in vivo complementation test in a malate dehydrogenase deleted Escherichia coli strain. |
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