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Molecules 2018, 23(1), 184; https://doi.org/10.3390/molecules23010184

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

1
Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands
2
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 Utrecht, The Netherlands
3
Netherlands Proteomics Center, Padualaan 8, 3584 Utrecht, The Netherlands
*
Author to whom correspondence should be addressed.
Received: 8 December 2017 / Revised: 11 January 2018 / Accepted: 14 January 2018 / Published: 16 January 2018
(This article belongs to the Special Issue Flavoenzymes)
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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 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. View Full-Text
Keywords: flavoprotein; proline dehydrogenase; protein engineering; protein oligomerization; solubility tag; suicide inhibition; TIM-barrel flavoprotein; proline dehydrogenase; protein engineering; protein oligomerization; solubility tag; suicide inhibition; TIM-barrel
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Huijbers, M.M.E.; van Alen, I.; Wu, J.W.; Barendregt, A.; Heck, A.J.R.; van Berkel, W.J.H. Functional Impact of the N-terminal Arm of Proline Dehydrogenase from Thermus thermophilus. Molecules 2018, 23, 184.

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