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Article

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.
Molecules 2018, 23(1), 184; https://doi.org/10.3390/molecules23010184
Received: 8 December 2017 / Revised: 11 January 2018 / Accepted: 14 January 2018 / Published: 16 January 2018
(This article belongs to the Special Issue Flavoenzymes)
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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MDPI and ACS Style

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. https://doi.org/10.3390/molecules23010184

AMA Style

Huijbers MME, Van Alen I, Wu JW, Barendregt A, Heck AJR, Van Berkel WJH. Functional Impact of the N-terminal Arm of Proline Dehydrogenase from Thermus thermophilus. Molecules. 2018; 23(1):184. https://doi.org/10.3390/molecules23010184

Chicago/Turabian Style

Huijbers, Mieke M.E., Ilona Van Alen, Jenny W. Wu, Arjan Barendregt, Albert J.R. Heck, and Willem J.H. Van Berkel 2018. "Functional Impact of the N-terminal Arm of Proline Dehydrogenase from Thermus thermophilus" Molecules 23, no. 1: 184. https://doi.org/10.3390/molecules23010184

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