|Title||Dimerization of Proline Dehydrogenase from Thermus thermophilus Is Crucial for Its Thermostability|
|Author(s)||Huijbers, Mieke M.E.; Wu, Jenny W.; Westphal, Adrie H.; Berkel, Willem J.H. van|
|Source||Biotechnology Journal 14 (2019)5. - ISSN 1860-6768|
|Publication type||Refereed Article in a scientific journal|
|Keyword(s)||flavoprotein - protein oligomerization - thermostability - Thermus thermophilus - triosephosphate isomerase barrel|
Thermus thermophilus proline dehydrogenase (TtProDH) catalyzes the first step in proline catabolism. The thermostable flavoenzyme consists of a distorted triosephosphate isomerase (TIM) barrel and three N-terminal helices: αA, αB, and αC. Using maltose-binding protein (MBP) fused constructs, it has been recently demonstrated that helix αC is crucial for TtProDH catalysis and for tetramerization through positioning of helix α8. Here, the structural features that determine the thermostability of TtProDH are reported. Selective disruption of two ion pairs in the dimerization interface of several MBP-TtProDH variants result in the formation of monomers. The newly created monomers have improved catalytic properties but their melting temperatures are decreased by more than 20 °C. Sequence comparison suggests that one of the ion-pairs involved in dimerization is unique for ProDHs from Thermus species. In summary, intermolecular ion-pairs improve the thermostability of TtProDH and a trade-off is made between thermostability and catalytic activity.