Functional Impact of the N-terminal Arm of Proline Dehydrogenase from Thermus thermophilus

• Main Authors: Mieke M. E. Huijbers, Ilona van Alen, Jenny W. Wu, Arjan Barendregt, Albert J. R. Heck, Willem J. H. van Berkel
• Format: Article
• Language: English
• Published: MDPI AG 2018-01-01
• Series: Molecules
• Subjects: flavoprotein; proline dehydrogenase; protein engineering; protein oligomerization; solubility tag; suicide inhibition; TIM-barrel
• Online Access: http://www.mdpi.com/1420-3049/23/1/184
• ISSN: 1420-3049

Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme that catalyzes the oxidation of proline to Δ1-pyrroline-5-carboxylate. Thermus thermophilus ProDH (TtProDH) contains in addition to its flavin-binding domain an N-terminal arm, consisting of helices αA, αB, and αC. Here, we report the biochemical properties of the helical arm truncated TtProDH variants ΔA, ΔAB, and ΔABC, produced with maltose-binding protein as solubility tag. All three truncated variants show similar spectral properties as TtProDH, indicative of a conserved flavin-binding pocket. ΔA and ΔAB are highly active tetramers that rapidly react with the suicide inhibitor N-propargylglycine. Removal of the entire N-terminal arm (ΔABC) results in barely active dimers that are incapable of forming a flavin adduct with N-propargylglycine. Characterization of V32D, Y35F, and V36D variants of ΔAB established that a hydrophobic patch between helix αC and helix α8 is critical for TtProDH catalysis and tetramer stabilization.