Interpreting the Plastid Carbon, Nitrogen, and Energy Status. A Role for PII?

Moorhead, Greg B. G.; Smith, Catherine Delano

doi:10.1104/pp.103.025627

Cited by 54 publications

(35 citation statements)

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“…Although PII from Arabidopsis thaliana, encoded by GLB1, has been identified and biochemically characterized, the physiological role of PII proteins in plants remains elusive (17,26,37). As it is in cyanobacteria, transcription of GLB1 is regulated by light and carbon-nitrogen status (12,17,22), in agreement with a role in coordination of photosynthesis and nitrogen assimilation.…”

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Interactions between the Nitrogen Signal Transduction Protein PII and N -Acetyl Glutamate Kinase in Organisms That Perform Oxygenic Photosynthesis

et al. 2004

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PII, one of the most conserved signal transduction proteins, is believed to be a key player in the coordination of nitrogen assimilation and carbon metabolism in bacteria, archaea, and plants. However, the identity of PII receptors remains elusive, particularly in photosynthetic organisms. Here we used yeast two-hybrid approaches to identify new PII receptors and to explore the extent of conservation of PII signaling mechanisms between eubacteria and photosynthetic eukaryotes. Screening of Synechococcus sp. strain PCC 7942 libraries with PII as bait resulted in identification of N-acetyl glutamate kinase (NAGK), a key enzyme in the biosynthesis of arginine. The integrity of Ser49, a residue conserved in PII proteins from organisms that perform oxygenic photosynthesis, appears to be essential for NAGK binding. The effect of glnB mutations on NAGK activity is consistent with positive regulation of NAGK by PII. Phylogenetic and yeast two-hybrid analyses strongly suggest that there was conservation of the NAGK-PII regulatory interaction in the evolution of cyanobacteria and chloroplasts, providing insight into the function of eukaryotic PII-like proteins.PII, one of the most conserved and widespread nitrogen signal transduction proteins, is an important player in the coordination of nitrogen assimilation and carbon metabolism (1,8,27). In the enteric system, two paralogous genes, glnB and glnK, encode PII proteins. Their receptors include converter enzymes (uridylyltranferase/uridylyl-removing enzyme or GlnD) and downstream targets (NtrB, the histidine kinase of the NtrB-NtrC two-component system, adenylyltransferase or GlnE, and the membrane-bound ammonium transporter AmtB). In cyanobacteria, a unique PII protein (referred to as GlnB), encoded by the glnB gene, is regulated by separate kinase and phosphatase activities (11); one of these activities, PphA, a PP2C-type phosphatase, has recently been identified in Synechocystis sp. strain PCC 6803 (18) as the PII phosphatase. In Synechococcus sp. strain PCC 7942, involvement of PII in the short-term ammonium inhibition of nitrate uptake has been well established (21), but direct protein-protein interactions between transport components and PII have not been reported.Eukaryotic PII-encoding genes were first found in the chloroplast of the red alga Porphyra purpurea (31), and they seem to be present in a wide variety of higher plants, encoding proteins with high levels of homology to cyanobacterial PII proteins. Although PII from Arabidopsis thaliana, encoded by GLB1, has been identified and biochemically characterized, the physiological role of PII proteins in plants remains elusive (17, 26, 37). As it is in cyanobacteria, transcription of GLB1 is regulated by light and carbon-nitrogen status (12,17,22), in agreement with a role in coordination of photosynthesis and nitrogen assimilation. Given the common evolutionary origin of PII proteins in organisms that perform oxygenic photosynthesis, it seems likely that mechanisms and components of the corresponding signal tra...

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confidence: 94%

Interactions between the Nitrogen Signal Transduction Protein PII and N -Acetyl Glutamate Kinase in Organisms That Perform Oxygenic Photosynthesis

et al. 2004

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“…Cyanobacteria have one P II homologue of the GlnB subfamily; its structure and ligand binding properties is highly similar to that of the E. coli P II proteins as shown for the unicellular cyanobacterium Synechocococcus elongatus strain PCC 7942 (20 -22). P II proteins from plant chloroplasts are closely related to the cyanobacterial homologues and display similar ligand binding properties (23). Generally, cyanobacterial P II proteins are subject to covalent modification in response to the cellular nitrogen/carbon status through phosphorylation at seryl residue 49, as shown for S. elongatus P II (4,24).…”

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confidence: 99%

Complex Formation and Catalytic Activation by the PII Signaling Protein of N-Acetyl-l-glutamate Kinase from Synechococcus elongatus Strain PCC 7942

Mani

Urbanke

Forchhammer

2004

Journal of Biological Chemistry

141

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The signal transduction protein P II from the cyanobacterium Synechococcus elongatus strain PCC 7942 forms a complex with the key enzyme of arginine biosynthesis, N-acetyl-L-glutamate kinase (NAGK). Here we report the effect of complex formation on the catalytic properties of NAGK. Although pH and ion dependence are not affected, the catalytic efficiency of NAGK is strongly enhanced by binding of P II , with K m decreasing by a factor of 10 and V max increasing 4-fold. In addition, arginine feedback inhibition of NAGK is strongly decreased in the presence of P II , resulting in a tight control of NAGK activity under physiological conditions by P II . Analysis of the NAGK-P II complex suggests that one P II trimer binds to one NAGK hexamer with a K d of ϳ3 nM. Complex formation is strongly affected by ATP and ADP. ADP is a strong inhibitor of complex formation, whereas ATP inhibits complex formation only in the absence of divalent cations or in the presence of Mg 2؉ ions, together with increased 2-oxoglutarate concentrations. Ca 2؉ is able to antagonize the negative effect of ATP and 2-oxoglutarate. ADP and ATP exert their adverse effect on NAGK-P II complex formation through binding to the P II protein.P II proteins constitute a large family of signal transduction proteins reported from all three domains of life. Bacterial P II signaling proteins have been shown to play pivotal roles in the control of various aspects of nitrogen assimilation, such as ammonium assimilation (reviewed in Refs. 1 and 2), uptake of nitrogen sources (3, 4), or nitrogen fixation (5). In these cellular processes, P II proteins exert control on the level of transcription, e.g. by interaction with a two-component sensor kinase NtrB (6) or with the NifL/NifA system (7). P II also exerts control on the level of enzyme activity, e.g. by modulating the activity of regulatory enzymes such as glutamine synthetase adenylyltransferase (8), the nitrogenase switch-off system DraT/DraG (9, 10), or the activity of transport systems (11). Interaction of the P II proteins with its targets of regulation, the P II receptors, has been studied in depth with the P II receptors in Escherichia coli, NtrB and GlnE (8,12,13), as well for NifL in nitrogen-fixing proteobacteria (7,14). In each case, the signal input status of the P II proteins plays a crucial role for the interaction. In proteobacteria, which typically encode two P II paralogues, GlnB and GlnK, the trimeric P II proteins are subject to covalent modification at tyrosyl residue 51 (15-17), located on the solvent-exposed T-loop, in response to the cellular nitrogen status that is perceived by the cellular glutamine level (18). In addition, P II proteins bind the effector molecules 2-oxoglutarate and ATP in a synergistic manner (19,20). Cyanobacteria have one P II homologue of the GlnB subfamily; its structure and ligand binding properties is highly similar to that of the E. coli P II proteins as shown for the unicellular cyanobacterium Synechocococcus elongatus strain PCC 7942 (20 -22). P II protein...

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“…Originally discovered as a factor necessary for the inactivation of Escherichia coli glutamine synthetase, PII is now known to play roles in the regulation of gene transcription, enzyme activity, and membrane channel function, all in response to cellular carbon, nitrogen, and energy status (3)(4)(5)(6). PII interprets the metabolic status of the cell by directly binding to ATP and 2-ketoglutarate, and in certain bacteria, nitrogen status is sensed via covalent modification of PII.…”

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Structural Basis for the Regulation of N-Acetylglutamate Kinase by PII in Arabidopsis thaliana

Mizuno

Moorhead

2007

Journal of Biological Chemistry

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PII is a highly conserved regulatory protein found in organisms across the three domains of life. In cyanobacteria and plants, PII relieves the feedback inhibition of the rate-limiting step in arginine biosynthesis catalyzed by N-acetylglutamate kinase (NAGK). To understand the molecular structural basis of enzyme regulation by PII, we have determined a 2.5-Å resolution crystal structure of a complex formed between two homotrimers of PII and a single hexamer of NAGK from Arabidopsis thaliana bound to the metabolites N-acetylglutamate, ADP, ATP, and arginine. In PII, the T-loop and Trp 22 at the start of the ␣1-helix, which are both adjacent to the ATP-binding site of PII, contact two ␤-strands as well as the ends of two central helices (␣E and ␣G) in NAGK, the opposing ends of which form major portions of the ATP and N-acetylglutamate substratebinding sites. The binding of Mg 2؉ ⅐ATP to PII stabilizes a conformation of the T-loop that favors interactions with both open and closed conformations of NAGK. Interactions between PII and NAGK appear to limit the degree of opening and closing of the active-site cleft in opposition to a domain-separating inhibitory effect exerted by arginine, thus explaining the stimulatory effect of PII on the kinetics of arginine-inhibited NAGK.PII (GlnB) is now recognized as one of the most ancient and conserved signal transduction proteins known, with orthologs spread across the three domains of life (1, 2). Originally discovered as a factor necessary for the inactivation of Escherichia coli glutamine synthetase, PII is now known to play roles in the regulation of gene transcription, enzyme activity, and membrane channel function, all in response to cellular carbon, nitrogen, and energy status (3-6). PII interprets the metabolic status of the cell by directly binding to ATP and 2-ketoglutarate, and in certain bacteria, nitrogen status is sensed via covalent modification of PII. PII then directly interacts with a variety of proteins to regulate their function in response to the detected metabolic conditions.A eukaryotic PII protein has been discovered in several algae and higher plants. Arabidopsis thaliana PII is Ͼ50% identical to proteobacterial and cyanobacterial PII (7), but unlike these orthologs, it does not appear to be regulated by phosphorylation or uridylylation in response to nitrogen metabolites (8). Plant PII has a conserved chloroplast transit peptide that is cleaved upon entry into the chloroplast, where PII performs its function. PII knock-out plants display altered carbon and nitrogen metabolite levels as well as increased sensitivity to nitrite (9).To date, the sole interacting protein discovered for plant PII is the chloroplast enzyme N-acetylglutamate kinase (NAGK), 2 which catalyzes the second and rate-limiting step in the pathway of arginine biosynthesis (10). We (11) and others (12) have performed enzyme kinetics to define the role of PII in NAGK activity: although PII activates NAGK slightly, the primary purpose of binding appears to be relief of feedback inhi...

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Interpreting the Plastid Carbon, Nitrogen, and Energy Status. A Role for PII?

Cited by 54 publications

References 47 publications

Interactions between the Nitrogen Signal Transduction Protein PII and N -Acetyl Glutamate Kinase in Organisms That Perform Oxygenic Photosynthesis

Interactions between the Nitrogen Signal Transduction Protein PII and N -Acetyl Glutamate Kinase in Organisms That Perform Oxygenic Photosynthesis

Complex Formation and Catalytic Activation by the PII Signaling Protein of N-Acetyl-l-glutamate Kinase from Synechococcus elongatus Strain PCC 7942

Structural Basis for the Regulation of N-Acetylglutamate Kinase by PII in Arabidopsis thaliana

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