Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from <i>Escherichia coli</i> 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 <i>Escherichia coli</i> GAPDH) in two alternative conformations (I and II). It is uncertain what triggers this loop rear...

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Autores principales:	Li Zhang, Meiruo Liu, Luyao Bao, Kristina I. Boström, Yucheng Yao, Jixi Li, Shaohua Gu, Chaoneng Ji
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	<i>Escherichia coli</i> type 1 GAPDH 1,3-diphosphoglycerate BPG thioacyl intermediate structure catalytic process Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/61d9c95b9c3c444bbfb3e5553c702d76
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spelling	oai:doaj.org-article:61d9c95b9c3c444bbfb3e5553c702d762021-11-25T16:51:55ZNovel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from <i>Escherichia coli</i> Provide New Insights into the Mechanism of Generation of 1,3-Bisphosphoglyceric Acid10.3390/biom111115652218-273Xhttps://doaj.org/article/61d9c95b9c3c444bbfb3e5553c702d762021-10-01T00:00:00Zhttps://www.mdpi.com/2218-273X/11/11/1565https://doaj.org/toc/2218-273XGlyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved enzyme involved in the ubiquitous process of glycolysis and presents a loop (residues 208–215 of <i>Escherichia coli</i> 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.Li ZhangMeiruo LiuLuyao BaoKristina I. BoströmYucheng YaoJixi LiShaohua GuChaoneng JiMDPI AGarticle<i>Escherichia coli</i> type 1 GAPDH1,3-diphosphoglycerateBPGthioacyl intermediate structurecatalytic processMicrobiologyQR1-502ENBiomolecules, Vol 11, Iss 1565, p 1565 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	<i>Escherichia coli</i> type 1 GAPDH 1,3-diphosphoglycerate BPG thioacyl intermediate structure catalytic process Microbiology QR1-502
spellingShingle	<i>Escherichia coli</i> type 1 GAPDH 1,3-diphosphoglycerate BPG thioacyl intermediate structure catalytic process Microbiology QR1-502 Li Zhang Meiruo Liu Luyao Bao Kristina I. Boström Yucheng Yao Jixi Li Shaohua Gu Chaoneng Ji Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from <i>Escherichia coli</i> Provide New Insights into the Mechanism of Generation of 1,3-Bisphosphoglyceric Acid
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 <i>Escherichia coli</i> 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.
format	article
author	Li Zhang Meiruo Liu Luyao Bao Kristina I. Boström Yucheng Yao Jixi Li Shaohua Gu Chaoneng Ji
author_facet	Li Zhang Meiruo Liu Luyao Bao Kristina I. Boström Yucheng Yao Jixi Li Shaohua Gu Chaoneng Ji
author_sort	Li Zhang
title	Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from <i>Escherichia coli</i> 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 <i>Escherichia coli</i> 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 <i>Escherichia coli</i> 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 <i>Escherichia coli</i> 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 <i>Escherichia coli</i> 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 <i>escherichia coli</i> provide new insights into the mechanism of generation of 1,3-bisphosphoglyceric acid
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/61d9c95b9c3c444bbfb3e5553c702d76
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Novel Structures of Type 1 Glyceraldehyde-3-phosphate Dehydrogenase from <i>Escherichia coli</i> Provide New Insights into the Mechanism of Generation of 1,3-Bisphosphoglyceric Acid

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