Molecular Identification and Characterization of Cytosolic Isoforms of Glutamine Synthetase in Maize Roots

Sakakibara, Hitoshi; Shimizu, Hitoshi; Hase, Toshiharu; Yamazaki, Y.; Takao, Toshifumi; Shimonishi, Yasutsugu; Sugiyama, Tatsuo

doi:10.1074/jbc.271.47.29561

Cited by 69 publications

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“…After uptake of nitrate, plants first reduce it to ammonia, and then assimilate it into an organic compound as an amide moiety of glutamine. Because glutamine synthetase (GS) 7 catalyzes the very step of assimilation of inorganic nitrogen and because the amide moiety of glutamine is utilized as the donor of amino residue to synthesize a number of essential metabolites such as amino acids, nucleic acids, and amino sugars, glutamine synthesis by plant GS is the cornerstone of plant productivity and thus nitrogen nourishment of all animals on the Earth. For this reason, the importance of plant GS is comparable with that of ribulose-1,5-bisphosphate carboxylase/oxygenase, the carbon dioxide assimilating enzyme (1).…”

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“…GS1 is encoded by a small multigene family, and the GS1 members are further categorized into two groups based on expression profile in response to external nitrogen status, enzymatic property, and physicochemical stability. Maize has five GS1s (GS1a-GS1e) (6,7), and two representative isoforms that are well characterized namely GS1a and GS1d, show high sequence identity (86%), but show remarkable difference in stability (7). We chose GS1a for an initial trial for three-dimensional structure determination because of its high stability (7).…”

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“…The atomic coordinates and structure factors (code 2D3A, 2D3B, and 2D3C) 81-6-6879-8636; E-mail: kusunoki@protein.osaka-u.ac.jp. 7 The abbreviations used are: GS, glutamine synthetase; MetSox-P, methionine sulfoximine phosphate; PEG, polyethylene glycol; MetSox, methionine sulfoximine; PPT-P, phosphinothricin phosphate; AMPPNP, adenylyl imidodiphosphate; PPT, phosphinothricin; NCS, non-crystallographic symmetry. …”

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“…Maize has five GS1s (GS1a-GS1e) (6,7), and two representative isoforms that are well characterized namely GS1a and GS1d, show high sequence identity (86%), but show remarkable difference in stability (7). We chose GS1a for an initial trial for three-dimensional structure determination because of its high stability (7). We report the role of Ile-161 responsible for differences in heat stability between the two GS isozymes, and three complex structures of maize GS1a revealing the reaction mechanism of phosphate transfer and inhibitory mechanism of PPT used as a herbicide.…”

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Atomic Structure of Plant Glutamine Synthetase

Unno

Uchida

Sugawara

et al. 2006

Journal of Biological Chemistry

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138

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Plants provide nourishment for animals and other heterotrophs as the sole primary producer in the food chain. Glutamine synthetase (GS), one of the essential enzymes for plant autotrophy catalyzes the incorporation of ammonia into glutamate to generate glutamine with concomitant hydrolysis of ATP, and plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Elucidation of the atomic structure of higher plant GS is important to understand its detailed reaction mechanism and to obtain further insight into plant productivity and agronomical utility. Here we report the first crystal structures of maize (Zea mays L.) GS. The structure reveals a unique decameric structure that differs significantly from the bacterial GS structure. Higher plants have several isoenzymes of GS differing in heat stability and catalytic properties for efficient responses to variation in the environment and nutrition. A key residue responsible for the heat stability was found to be Ile-161 in GS1a. The three structures in complex with substrate analogues, including phosphinothricin, a widely used herbicide, lead us to propose a mechanism for the transfer of phosphate from ATP to glutamate and to interpret the inhibitory action of phosphinothricin as a guide for the development of new potential herbicides.Inorganic nitrogen is an essential, often limiting nutrient for plant growth and development. In most natural soils, nitrate is the major form of inorganic nitrogen. After uptake of nitrate, plants first reduce it to ammonia, and then assimilate it into an organic compound as an amide moiety of glutamine. Because glutamine synthetase (GS) 7 catalyzes the very step of assimilation of inorganic nitrogen and because the amide moiety of glutamine is utilized as the donor of amino residue to synthesize a number of essential metabolites such as amino acids, nucleic acids, and amino sugars, glutamine synthesis by plant GS is the cornerstone of plant productivity and thus nitrogen nourishment of all animals on the Earth. For this reason, the importance of plant GS is comparable with that of ribulose-1,5-bisphosphate carboxylase/oxygenase, the carbon dioxide assimilating enzyme (1).Comparison of the primary structures of GSs from prokaryotes and eukaryotes, results in plant GS being categorized as type II, this type commonly occurring in eukaryotes including animals (2). In contrast, type I GS is widely found in prokaryotes (2). The regulatory mechanisms of type I GS activity such as adenylylation and metabolite feedback have been thoroughly characterized (3). The crystal structures of GS from Mycobacterium tuberculosis (4) and Salmonella typhimurium (5) have been determined and the proteins shown to be dodecameric, with each dodecamer being composed of one identical subunit with a molecular mass of about 52 kDa. Types I and II GSs are thought to share a common ancestor but to have diverged into the two types at a very early stage during molecula...

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Atomic Structure of Plant Glutamine Synthetase

Unno

Uchida

Sugawara

et al. 2006

Journal of Biological Chemistry

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138

195

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“…Since then, advances in recombinant DNA technology and the advent of affinity purification technologies have greatly facilitated the isolation of GS material suitable for crystallization. The first crystal structure of a GSII enzyme was of the Zea mays GS1a protein [7], which was heterologously expressed in Escherichia coli and purified using a combination of anion exchange and affinity chromatography steps [8]. More recently, several structures of GSII enzymes purified with affinity tags [9,10] have been reported.…”

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Crystallization of recombinant Bacteroides fragilis glutamine synthetase (GlnN) isolated using a novel and rapid purification protocol

Rooyen¹,

Abratt²,

Belrhali³

et al. 2010

Protein Expression and Purification

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a b s t r a c tGlutamine synthetase enzymes (GSs) are large oligomeric enzymes that play a critical role in nitrogen metabolism in all forms of life. To date, no crystal structures exist for the family of large ($1 MDa) type III GS enzymes, which only share 9% sequence identity with the well characterized GSI and GSII enzymes. Here we present a novel protocol for the isolation of untagged Bacteroides fragilis GlnN expressed in an auxotrophic Escherichia coli strain. The rapid and scalable two-step protocol utilized differential precipitation by divalent cations followed by affinity chromatography to produce suitable quantities of homogenous material for structural characterization. Subsequent optimizations to the sample stability and solubility led to the discovery of conditions for the production of the first diffraction quality crystals of a type III GS enzyme.Ó 2010 Elsevier Inc. All rights reserved. IntroductionGlutamine synthetases (GSs) 1 are large oligomeric enzymes that play a central role in nitrogen assimilation, catalysing the condensation of ammonium and glutamate to form glutamine, a precursor for the synthesis of many critical bio-molecules. The ancient [1] and ubiquitous [2] GS superfamily is, therefore, evolutionarily diverse and can be divided into three main groupings, namely GSI, GSII and GSIII, on the basis of amino-acid divergence [3].GSI and GSII enzymes have been purified from numerous bacterial and eukaryotic sources, respectively, over the past five decades of biochemical and structural investigations into their functioning and regulation [4]. The first crystal structure of a GSI enzyme [5] was solved using material prepared from an adenylylation deficient Salmonella typhimurium strain using a combination of simple differential precipitation techniques and nucleotide-analogue affinity chromatography [6]. Since then, advances in recombinant DNA technology and the advent of affinity purification technologies have greatly facilitated the isolation of GS material suitable for crystallization. The first crystal structure of a GSII enzyme was of the Zea mays GS1a protein [7], which was heterologously expressed in Escherichia coli and purified using a combination of anion exchange and affinity chromatography steps [8]. More recently, several structures of GSII enzymes purified with affinity tags [9,10] have been reported.In comparison, GSIII enzymes have only been isolated and characterized from a small number of sources [11][12][13] despite the widespread occurrence of homologues in a number of evolutionarily divergent organisms [14][15][16], including, the anaerobic pathogenic protozoan Trichomonas vaginalis [17]. To date, the only structural information describing the large GSIII enzymes ($1 MDa), which share only $9% sequence identity with the GSI enzymes, is from a low-resolution cryo-EM reconstruction of the GlnN enzyme from the opportunistic human pathogen Bacteroides fragilis [3]. A detailed understanding of the functioning and evolution of these divergent enzymes in light of the known ...

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Nitrogen Assimilation and its Relevance to Crop Improvement

Lea

Miflin

2018

Annual Plant Reviews Online

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The majority if not all of the organic nitrogen in plants is derived from the assimilation of ammonia into the amide position of glutamine by the enzyme glutamine synthetase (GS). A second enzyme, glutamate synthase, also known as glutamine:2‐oxoglutarate amidotransferase (GOGAT), carries out the transfer of the amide group of glutamine to 2‐oxoglutarate to yield two molecules of glutamate and thus completes the assimilation of ammonia into amino acids. This GS/GOGAT pathway of ammonia assimilation is of crucial importance for crop growth and productivity and ultimately animal and human nutrition. Glutamate dehydrogenase (GDH) is now considered to be only involved in glutamate catabolism to form ammonia, an important role in N recycling within the plant. Nitrogen is also often diverted from glutamine to asparagine as a temporary measure during periods of carbohydrate shortage and excess of reduced nitrogen. This diversion requires the action of asparagine synthetase in the anabolic reaction and asparaginase in the catabolic reaction. This chapter describes the properties of these enzymes in the assimilation and reassimilation of nitrogen and in particular the genes that encode them, their complexity and the time and place they are expressed. Plant transformation has allowed the construction of a range of plants with enhanced and decreased activity of several of these enzymes, some of which have shown improved agronomic performance.

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Molecular Identification and Characterization of Cytosolic Isoforms of Glutamine Synthetase in Maize Roots

Cited by 69 publications

References 36 publications

Atomic Structure of Plant Glutamine Synthetase

Atomic Structure of Plant Glutamine Synthetase

Crystallization of recombinant Bacteroides fragilis glutamine synthetase (GlnN) isolated using a novel and rapid purification protocol

Nitrogen Assimilation and its Relevance to Crop Improvement

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