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Structural Studies of Glutamate Dehydrogenase (Isoform 1) From Arabidopsis thaliana, an Important Enzyme at the Branch-Point Between Carbon and Nitrogen Metabolism

Glutamate dehydrogenase (GDH) releases ammonia in a reversible NAD(P)(+)-dependent oxidative deamination of glutamate that yields 2-oxoglutarate (2OG). In current perception, GDH contributes to Glu homeostasis and plays a significant role at the junction of carbon and nitrogen assimilation pathways....

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Autores principales: Grzechowiak, Marta, Sliwiak, Joanna, Jaskolski, Mariusz, Ruszkowski, Milosz
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326016/
https://www.ncbi.nlm.nih.gov/pubmed/32655590
http://dx.doi.org/10.3389/fpls.2020.00754
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author Grzechowiak, Marta
Sliwiak, Joanna
Jaskolski, Mariusz
Ruszkowski, Milosz
author_facet Grzechowiak, Marta
Sliwiak, Joanna
Jaskolski, Mariusz
Ruszkowski, Milosz
author_sort Grzechowiak, Marta
collection PubMed
description Glutamate dehydrogenase (GDH) releases ammonia in a reversible NAD(P)(+)-dependent oxidative deamination of glutamate that yields 2-oxoglutarate (2OG). In current perception, GDH contributes to Glu homeostasis and plays a significant role at the junction of carbon and nitrogen assimilation pathways. GDHs are members of a superfamily of ELFV (Glu/Leu/Phe/Val) amino acid dehydrogenases and are subdivided into three subclasses, based on coenzyme specificity: NAD(+)-specific, NAD(+)/NADP(+) dual-specific, and NADP(+)-specific. We determined in this work that the mitochondrial AtGDH1 isozyme from A. thaliana is NAD(+)-specific. Altogether, A. thaliana expresses three GDH isozymes (AtGDH1-3) targeted to mitochondria, of which AtGDH2 has an extra EF-hand motif and is stimulated by calcium. Our enzymatic assays of AtGDH1 established that its sensitivity to calcium is negligible. In vivo the AtGDH1-3 enzymes form homo- and heterohexamers of varied composition. We solved the crystal structure of recombinant AtGDH1 in the apo-form and in complex with NAD(+) at 2.59 and 2.03 Å resolution, respectively. We demonstrate also that both in the apo form and in 1:1 complex with NAD(+), it forms D(3)-symmetric homohexamers. A subunit of AtGDH1 consists of domain I, which is involved in hexamer formation and substrate binding, and of domain II which binds coenzyme. Most of the subunits in our crystal structures, including those in NAD(+) complex, are in open conformation, with domain II forming a large (albeit variable) angle with domain I. One of the subunits of the AtGDH1-NAD(+) hexamer contains a serendipitous 2OG molecule in the active site, causing a dramatic (∼25°) closure of the domains. We provide convincing evidence that the N-terminal peptide preceding domain I is a mitochondrial targeting signal, with a predicted cleavage site for mitochondrial processing peptidase (MPP) at Leu17-Leu18 that is followed by an unexpected potassium coordination site (Ser27, Ile30). We also identified several MPD [(+/-)-2-methyl-2,4-pentanediol] binding sites with conserved sequence. Although AtGDH1 is insensitive to MPD in our assays, the observation of druggable sites opens a potential for non-competitive herbicide design.
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spelling pubmed-73260162020-07-09 Structural Studies of Glutamate Dehydrogenase (Isoform 1) From Arabidopsis thaliana, an Important Enzyme at the Branch-Point Between Carbon and Nitrogen Metabolism Grzechowiak, Marta Sliwiak, Joanna Jaskolski, Mariusz Ruszkowski, Milosz Front Plant Sci Plant Science Glutamate dehydrogenase (GDH) releases ammonia in a reversible NAD(P)(+)-dependent oxidative deamination of glutamate that yields 2-oxoglutarate (2OG). In current perception, GDH contributes to Glu homeostasis and plays a significant role at the junction of carbon and nitrogen assimilation pathways. GDHs are members of a superfamily of ELFV (Glu/Leu/Phe/Val) amino acid dehydrogenases and are subdivided into three subclasses, based on coenzyme specificity: NAD(+)-specific, NAD(+)/NADP(+) dual-specific, and NADP(+)-specific. We determined in this work that the mitochondrial AtGDH1 isozyme from A. thaliana is NAD(+)-specific. Altogether, A. thaliana expresses three GDH isozymes (AtGDH1-3) targeted to mitochondria, of which AtGDH2 has an extra EF-hand motif and is stimulated by calcium. Our enzymatic assays of AtGDH1 established that its sensitivity to calcium is negligible. In vivo the AtGDH1-3 enzymes form homo- and heterohexamers of varied composition. We solved the crystal structure of recombinant AtGDH1 in the apo-form and in complex with NAD(+) at 2.59 and 2.03 Å resolution, respectively. We demonstrate also that both in the apo form and in 1:1 complex with NAD(+), it forms D(3)-symmetric homohexamers. A subunit of AtGDH1 consists of domain I, which is involved in hexamer formation and substrate binding, and of domain II which binds coenzyme. Most of the subunits in our crystal structures, including those in NAD(+) complex, are in open conformation, with domain II forming a large (albeit variable) angle with domain I. One of the subunits of the AtGDH1-NAD(+) hexamer contains a serendipitous 2OG molecule in the active site, causing a dramatic (∼25°) closure of the domains. We provide convincing evidence that the N-terminal peptide preceding domain I is a mitochondrial targeting signal, with a predicted cleavage site for mitochondrial processing peptidase (MPP) at Leu17-Leu18 that is followed by an unexpected potassium coordination site (Ser27, Ile30). We also identified several MPD [(+/-)-2-methyl-2,4-pentanediol] binding sites with conserved sequence. Although AtGDH1 is insensitive to MPD in our assays, the observation of druggable sites opens a potential for non-competitive herbicide design. Frontiers Media S.A. 2020-06-05 /pmc/articles/PMC7326016/ /pubmed/32655590 http://dx.doi.org/10.3389/fpls.2020.00754 Text en Copyright © 2020 Grzechowiak, Sliwiak, Jaskolski and Ruszkowski. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
Grzechowiak, Marta
Sliwiak, Joanna
Jaskolski, Mariusz
Ruszkowski, Milosz
Structural Studies of Glutamate Dehydrogenase (Isoform 1) From Arabidopsis thaliana, an Important Enzyme at the Branch-Point Between Carbon and Nitrogen Metabolism
title Structural Studies of Glutamate Dehydrogenase (Isoform 1) From Arabidopsis thaliana, an Important Enzyme at the Branch-Point Between Carbon and Nitrogen Metabolism
title_full Structural Studies of Glutamate Dehydrogenase (Isoform 1) From Arabidopsis thaliana, an Important Enzyme at the Branch-Point Between Carbon and Nitrogen Metabolism
title_fullStr Structural Studies of Glutamate Dehydrogenase (Isoform 1) From Arabidopsis thaliana, an Important Enzyme at the Branch-Point Between Carbon and Nitrogen Metabolism
title_full_unstemmed Structural Studies of Glutamate Dehydrogenase (Isoform 1) From Arabidopsis thaliana, an Important Enzyme at the Branch-Point Between Carbon and Nitrogen Metabolism
title_short Structural Studies of Glutamate Dehydrogenase (Isoform 1) From Arabidopsis thaliana, an Important Enzyme at the Branch-Point Between Carbon and Nitrogen Metabolism
title_sort structural studies of glutamate dehydrogenase (isoform 1) from arabidopsis thaliana, an important enzyme at the branch-point between carbon and nitrogen metabolism
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326016/
https://www.ncbi.nlm.nih.gov/pubmed/32655590
http://dx.doi.org/10.3389/fpls.2020.00754
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