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Functional impact of the n-terminal arm of proline dehydrogenase from thermus thermophilus

By Mieke M.E. Huijbers, Ilona van Alen, Jenny W. Wu, Arjan Barendregt, Albert J.R. Heck and Willem J.H. Van Berkel


Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme that catalyzes the oxidation of proline to ∆1-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 ∆A, ∆AB, and ∆ABC, 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. ∆A and ∆AB are highly active tetramers that rapidly react with the suicide inhibitor N-propargylglycine. Removal of the entire N-terminal arm (∆ABC) results in barely active dimers that are incapable of forming a flavin adduct with N-propargylglycine. Characterization of V32D, Y35F, and V36D variants of ∆AB established that a hydrophobic patch between helix αC and helix α8 is critical for TtProDH catalysis and tetramer stabilization

Topics: Flavoprotein, Proline dehydrogenase, Protein engineering, Protein oligomerization, Solubility tag, Suicide inhibition, TIM-barrel
Year: 2018
DOI identifier: 10.3390/molecules23010184
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Provided by: NARCIS
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