کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
6456518 | 1420185 | 2016 | 7 صفحه PDF | دانلود رایگان |
- MBP-tagged TtProDH is an aggregation-prone homotetramer.
- Aggregation of TtProDH can be prevented by increasing the polarity of the N-terminal helix.
- The newly produced TtProDH variant has excellent catalytic properties.
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.
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Journal: Journal of Molecular Catalysis B: Enzymatic - Volume 134, Part B, December 2016, Pages 340-346