A more polar N-terminal helix releases MBP-tagged Thermus thermophilus proline dehydrogenase from tetramer-polymer self-association

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 FIDE/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. (C) 2016 Elsevier B.V. All rights reserved.