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Functional Impact of the N-terminal Arm of Proline Dehydrogenase from Thermus thermophilus
Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme that catalyzes the oxidation of proline to Δ(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 bioch...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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MDPI
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6017737/ https://www.ncbi.nlm.nih.gov/pubmed/29337919 http://dx.doi.org/10.3390/molecules23010184 |
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author | Huijbers, Mieke M. E. van Alen, Ilona Wu, Jenny W. Barendregt, Arjan Heck, Albert J. R. van Berkel, Willem J. H. |
author_facet | Huijbers, Mieke M. E. van Alen, Ilona Wu, Jenny W. Barendregt, Arjan Heck, Albert J. R. van Berkel, Willem J. H. |
author_sort | Huijbers, Mieke M. E. |
collection | PubMed |
description | Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme that catalyzes the oxidation of proline to Δ(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 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. |
format | Online Article Text |
id | pubmed-6017737 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-60177372018-11-13 Functional Impact of the N-terminal Arm of Proline Dehydrogenase from Thermus thermophilus Huijbers, Mieke M. E. van Alen, Ilona Wu, Jenny W. Barendregt, Arjan Heck, Albert J. R. van Berkel, Willem J. H. Molecules Article Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme that catalyzes the oxidation of proline to Δ(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 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. MDPI 2018-01-16 /pmc/articles/PMC6017737/ /pubmed/29337919 http://dx.doi.org/10.3390/molecules23010184 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Huijbers, Mieke M. E. van Alen, Ilona Wu, Jenny W. Barendregt, Arjan Heck, Albert J. R. van Berkel, Willem J. H. Functional Impact of the N-terminal Arm of Proline Dehydrogenase from Thermus thermophilus |
title | Functional Impact of the N-terminal Arm of Proline Dehydrogenase from Thermus thermophilus |
title_full | Functional Impact of the N-terminal Arm of Proline Dehydrogenase from Thermus thermophilus |
title_fullStr | Functional Impact of the N-terminal Arm of Proline Dehydrogenase from Thermus thermophilus |
title_full_unstemmed | Functional Impact of the N-terminal Arm of Proline Dehydrogenase from Thermus thermophilus |
title_short | Functional Impact of the N-terminal Arm of Proline Dehydrogenase from Thermus thermophilus |
title_sort | functional impact of the n-terminal arm of proline dehydrogenase from thermus thermophilus |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6017737/ https://www.ncbi.nlm.nih.gov/pubmed/29337919 http://dx.doi.org/10.3390/molecules23010184 |
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