|Title||A more polar N-terminal helix releases MBP-tagged Thermus thermophilus proline dehydrogenase from tetramer-polymer self-association|
|Author(s)||Huijbers, Mieke M.E.; Berkel, Willem J.H. van|
|Source||Journal of Molecular Catalysis. B, Enzymatic 134 (2016). - ISSN 1381-1177 - p. 340 - 346.|
|Publication type||Refereed Article in a scientific journal|
|Keyword(s)||Flavoprotein - Molecular self-association - Proline dehydrogenase - Protein oligomerization - Thermus thermophilus|
Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme involved in the biosynthesis of . l-glutamate. ProDH is of interest for biocatalysis because the protein might be applied in multi-enzyme reactions for the synthesis of structurally complex molecules. We recently demonstrated that the thermotolerant ProDH from . Thermus thermophilus (TtProDH) is overproduced in . Escherichia coli when using maltose-binding protein (MBP) as a solubility tag. However, MBP-TtProDH and MBP-clipped TtProDH are prone to aggregation through non-native self-association. Here we provide evidence that the hydrophobic N-terminal helix of TtProDH is responsible for the self-association process. The more polar MBP-tagged F10E/L12E variant exclusively forms tetramers and exhibits excellent catalytic features over a wide range of temperatures. Understanding the hydrodynamic and catalytic properties of thermostable enzymes is important for the development of industrial biocatalysts as well as for pharmaceutical applications.