1. Functional Impact of the N-terminal Arm of Proline Dehydrogenase from Thermus thermophilus.
- Author
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Huijbers, Mieke M. E., van Alen, Ilona, Wu, Jenny W., Barendregt, Arjan, Heck, Albert J. R., and van Berkel, Willem J. H.
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N-terminal residues , *PROLINE , *THERMUS thermophilus , *FLAVOPROTEINS , *OLIGOMERIZATION - Abstract
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 ΔA, ΔB, and ΔC. 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. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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