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Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from Escherichia coli Provide New Insights into the Mechanism of Generation of 1,3-Bisphosphoglyceric Acid

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved enzyme involved in the ubiquitous process of glycolysis and presents a loop (residues 208–215 of Escherichia coli GAPDH) in two alternative conformations (I and II). It is uncertain what triggers this loop rearrangement, as well...

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Autores principales: Zhang, Li, Liu, Meiruo, Bao, Luyao, Boström, Kristina I., Yao, Yucheng, Li, Jixi, Gu, Shaohua, Ji, Chaoneng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8615399/
https://www.ncbi.nlm.nih.gov/pubmed/34827563
http://dx.doi.org/10.3390/biom11111565
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author Zhang, Li
Liu, Meiruo
Bao, Luyao
Boström, Kristina I.
Yao, Yucheng
Li, Jixi
Gu, Shaohua
Ji, Chaoneng
author_facet Zhang, Li
Liu, Meiruo
Bao, Luyao
Boström, Kristina I.
Yao, Yucheng
Li, Jixi
Gu, Shaohua
Ji, Chaoneng
author_sort Zhang, Li
collection PubMed
description Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved enzyme involved in the ubiquitous process of glycolysis and presents a loop (residues 208–215 of Escherichia coli GAPDH) in two alternative conformations (I and II). It is uncertain what triggers this loop rearrangement, as well as which is the precise site from which phosphate attacks the thioacyl intermediate precursor of 1,3-bisphosphoglycerate (BPG). To clarify these uncertainties, we determined the crystal structures of complexes of wild-type GAPDH (WT) with NAD and phosphate or G3P, and of essentially inactive GAPDH mutants (C150S, H177A), trapping crystal structures for the thioacyl intermediate or for ternary complexes with NAD and either phosphate, BPG, or G3P. Analysis of these structures reported here lead us to propose that phosphate is located in the “new Pi site” attacks the thioester bond of the thioacyl intermediate to generate 1,3-bisphosphoglyceric acid (BPG). In the structure of the thioacyl intermediate, the mobile loop is in conformation II in subunits O, P, and R, while both conformations coexist in subunit Q. Moreover, only the Q subunit hosts bound NADH. In the R subunit, only the pyrophosphate part of NADH is well defined, and NADH is totally absent from the O and P subunits. Thus, the change in loop conformation appears to occur after NADH is produced, before NADH is released. In addition, two new D-glyceraldehyde-3-phosphate (G3P) binding forms are observed in WT.NAD.G3P and C150A+H177A.NAD.G3P. In summary, this paper improves our understanding of the GAPDH catalytic mechanism, particularly regarding BPG formation.
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spelling pubmed-86153992021-11-26 Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from Escherichia coli Provide New Insights into the Mechanism of Generation of 1,3-Bisphosphoglyceric Acid Zhang, Li Liu, Meiruo Bao, Luyao Boström, Kristina I. Yao, Yucheng Li, Jixi Gu, Shaohua Ji, Chaoneng Biomolecules Article Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved enzyme involved in the ubiquitous process of glycolysis and presents a loop (residues 208–215 of Escherichia coli GAPDH) in two alternative conformations (I and II). It is uncertain what triggers this loop rearrangement, as well as which is the precise site from which phosphate attacks the thioacyl intermediate precursor of 1,3-bisphosphoglycerate (BPG). To clarify these uncertainties, we determined the crystal structures of complexes of wild-type GAPDH (WT) with NAD and phosphate or G3P, and of essentially inactive GAPDH mutants (C150S, H177A), trapping crystal structures for the thioacyl intermediate or for ternary complexes with NAD and either phosphate, BPG, or G3P. Analysis of these structures reported here lead us to propose that phosphate is located in the “new Pi site” attacks the thioester bond of the thioacyl intermediate to generate 1,3-bisphosphoglyceric acid (BPG). In the structure of the thioacyl intermediate, the mobile loop is in conformation II in subunits O, P, and R, while both conformations coexist in subunit Q. Moreover, only the Q subunit hosts bound NADH. In the R subunit, only the pyrophosphate part of NADH is well defined, and NADH is totally absent from the O and P subunits. Thus, the change in loop conformation appears to occur after NADH is produced, before NADH is released. In addition, two new D-glyceraldehyde-3-phosphate (G3P) binding forms are observed in WT.NAD.G3P and C150A+H177A.NAD.G3P. In summary, this paper improves our understanding of the GAPDH catalytic mechanism, particularly regarding BPG formation. MDPI 2021-10-22 /pmc/articles/PMC8615399/ /pubmed/34827563 http://dx.doi.org/10.3390/biom11111565 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/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 (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Zhang, Li
Liu, Meiruo
Bao, Luyao
Boström, Kristina I.
Yao, Yucheng
Li, Jixi
Gu, Shaohua
Ji, Chaoneng
Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from Escherichia coli Provide New Insights into the Mechanism of Generation of 1,3-Bisphosphoglyceric Acid
title Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from Escherichia coli Provide New Insights into the Mechanism of Generation of 1,3-Bisphosphoglyceric Acid
title_full Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from Escherichia coli Provide New Insights into the Mechanism of Generation of 1,3-Bisphosphoglyceric Acid
title_fullStr Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from Escherichia coli Provide New Insights into the Mechanism of Generation of 1,3-Bisphosphoglyceric Acid
title_full_unstemmed Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from Escherichia coli Provide New Insights into the Mechanism of Generation of 1,3-Bisphosphoglyceric Acid
title_short Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from Escherichia coli Provide New Insights into the Mechanism of Generation of 1,3-Bisphosphoglyceric Acid
title_sort novel structures of type 1 glyceraldehyde-3-phosphate dehydrogenase from escherichia coli provide new insights into the mechanism of generation of 1,3-bisphosphoglyceric acid
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8615399/
https://www.ncbi.nlm.nih.gov/pubmed/34827563
http://dx.doi.org/10.3390/biom11111565
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