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Reconstitution of Escherichia coli glutamine synthetase adenylyltransferase from N-terminal and C-terminal fragments of the enzyme.

Jiang P, Ninfa AJ - Biochemistry (2009)

Bottom Line: Specifically, our results are consistent with the protein activators (PII and PII-UMP) binding to the enzyme domain with the opposing activity, with intramolecular signal transduction by direct interactions between the N-terminal AR catalytic domain and the C-terminal AT catalytic domain.Similarly, glutamine inhibition of the AR activity involved intramolecular signaling between the AT and AR domains.Finally, our results are consistent with the hypothesis that the AR activity of the N-terminal domain required activation by the opposing C-terminal (AT) domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0606, USA. pejiang@umich.edu

ABSTRACT
ATase brings about the short-term regulation of glutamine synthetase (GS) by catalyzing the adenylylation and deadenylylation of GS in response to signals of cellular nitrogen status and energy. The adenylyltransferase (AT) activity of ATase is activated by glutamine and by the unmodified form of the PII signal transduction protein and is inhibited by PII-UMP. Conversely, the adenylyl-removing (AR) activity of ATase is activated by PII-UMP and inhibited by unmodified PII and by glutamine. Here, we show that the enzyme can be reconstituted from two purified polypeptides that comprise the N-terminal two-thirds of the protein and the C-terminal one-third of the protein. Properties of the reconstituted enzyme support recent hypotheses for the sites of regulatory interactions and mechanisms for intramolecular signal transduction. Specifically, our results are consistent with the protein activators (PII and PII-UMP) binding to the enzyme domain with the opposing activity, with intramolecular signal transduction by direct interactions between the N-terminal AR catalytic domain and the C-terminal AT catalytic domain. Similarly, glutamine inhibition of the AR activity involved intramolecular signaling between the AT and AR domains. Finally, our results are consistent with the hypothesis that the AR activity of the N-terminal domain required activation by the opposing C-terminal (AT) domain.

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Reconstitution of the AR activity of ATase. (A) Schematic depiction of the domains comprising the ATC3 and ATN6 polypeptides. The numbers refer to the first and last amino acid residues in each species using the codon numbers for the wild-type full-length protein. (B) Nondenaturing 14% polyacrylamide gel electrophoresis of polypeptides in isolation and combination. Conditions for gel electrophoresis lacked any known regulators of ATase but included 1 mM MgCl2. Arrows indicate the position of the major ATN6 and ATC3 bands, as well as the position of the complex between these polypeptides. Each sample contained the indicated polypeptides at 6 μM each. Samples were incubated for 10 min at room temperature to allow the formation of complexes, prior to electrophoresis at 4 °C. (C) Reconstitution of the AR activity from the ATC3 and ATN6 polypeptides using an ATP-regenerating system to remove ADP. The initial rate of GS-AMP deadenylylation was measured as described in , with GS-AMP at 8 μM, PII-UMP at 5 μM, ATP at 1 mM, α-ketoglutarate at 1 mM, KPi at 1 mM, pyruvate kinase at 0.022 unit/μL, and PEP at 5 mM. (D) Reconstitution of the AR activity from the ATC3 and ATN6 polypeptides using the noncleavable analogue AMP-PNP in place of ATP. The initial rate of GS-AMP deadenylylation was measured as described in , with GS-AMP at 16 μM, PII-UMP at 5 μM, α-ketoglutarate at 1 mM, KPi at 1 mM, and AMP-PNP at 1 mM.
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fig2: Reconstitution of the AR activity of ATase. (A) Schematic depiction of the domains comprising the ATC3 and ATN6 polypeptides. The numbers refer to the first and last amino acid residues in each species using the codon numbers for the wild-type full-length protein. (B) Nondenaturing 14% polyacrylamide gel electrophoresis of polypeptides in isolation and combination. Conditions for gel electrophoresis lacked any known regulators of ATase but included 1 mM MgCl2. Arrows indicate the position of the major ATN6 and ATC3 bands, as well as the position of the complex between these polypeptides. Each sample contained the indicated polypeptides at 6 μM each. Samples were incubated for 10 min at room temperature to allow the formation of complexes, prior to electrophoresis at 4 °C. (C) Reconstitution of the AR activity from the ATC3 and ATN6 polypeptides using an ATP-regenerating system to remove ADP. The initial rate of GS-AMP deadenylylation was measured as described in , with GS-AMP at 8 μM, PII-UMP at 5 μM, ATP at 1 mM, α-ketoglutarate at 1 mM, KPi at 1 mM, pyruvate kinase at 0.022 unit/μL, and PEP at 5 mM. (D) Reconstitution of the AR activity from the ATC3 and ATN6 polypeptides using the noncleavable analogue AMP-PNP in place of ATP. The initial rate of GS-AMP deadenylylation was measured as described in , with GS-AMP at 16 μM, PII-UMP at 5 μM, α-ketoglutarate at 1 mM, KPi at 1 mM, and AMP-PNP at 1 mM.

Mentions: As a prelude to investigating this hypothesis, we tested whether different polypeptides derived from ATase could interact to produce the AR activity. That is, we examined whether the AR activity could be reconstituted from fragments of the ATase. Our earlier studies resulted in several polypeptides that contained various portions of the ATase (10); these were combined in all combinations (10). It was observed that in one case, a pair of polypeptides clearly associated with one another as observed by nondenaturing gel electrophoresis (10). This pair of polypeptides, called ATN6 and ATC3 (Figure 2A), when complexed together, results in the wild-type ATase missing one peptide bond, but in no case was AR activity obtained from combinations of polypeptides, including the case of the ATC3 and ATN6 pair (10). We will show here that our earlier failure to observe AR activity with this pair of polypeptides was due to the presence of ADP in the assay mixtures, and that in fact ATC3 and ATN6 polypeptides reconstitute the ATase, and their complex displays AR activity that is regulated by PII-UMP, PII, and glutamine (see ).


Reconstitution of Escherichia coli glutamine synthetase adenylyltransferase from N-terminal and C-terminal fragments of the enzyme.

Jiang P, Ninfa AJ - Biochemistry (2009)

Reconstitution of the AR activity of ATase. (A) Schematic depiction of the domains comprising the ATC3 and ATN6 polypeptides. The numbers refer to the first and last amino acid residues in each species using the codon numbers for the wild-type full-length protein. (B) Nondenaturing 14% polyacrylamide gel electrophoresis of polypeptides in isolation and combination. Conditions for gel electrophoresis lacked any known regulators of ATase but included 1 mM MgCl2. Arrows indicate the position of the major ATN6 and ATC3 bands, as well as the position of the complex between these polypeptides. Each sample contained the indicated polypeptides at 6 μM each. Samples were incubated for 10 min at room temperature to allow the formation of complexes, prior to electrophoresis at 4 °C. (C) Reconstitution of the AR activity from the ATC3 and ATN6 polypeptides using an ATP-regenerating system to remove ADP. The initial rate of GS-AMP deadenylylation was measured as described in , with GS-AMP at 8 μM, PII-UMP at 5 μM, ATP at 1 mM, α-ketoglutarate at 1 mM, KPi at 1 mM, pyruvate kinase at 0.022 unit/μL, and PEP at 5 mM. (D) Reconstitution of the AR activity from the ATC3 and ATN6 polypeptides using the noncleavable analogue AMP-PNP in place of ATP. The initial rate of GS-AMP deadenylylation was measured as described in , with GS-AMP at 16 μM, PII-UMP at 5 μM, α-ketoglutarate at 1 mM, KPi at 1 mM, and AMP-PNP at 1 mM.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

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fig2: Reconstitution of the AR activity of ATase. (A) Schematic depiction of the domains comprising the ATC3 and ATN6 polypeptides. The numbers refer to the first and last amino acid residues in each species using the codon numbers for the wild-type full-length protein. (B) Nondenaturing 14% polyacrylamide gel electrophoresis of polypeptides in isolation and combination. Conditions for gel electrophoresis lacked any known regulators of ATase but included 1 mM MgCl2. Arrows indicate the position of the major ATN6 and ATC3 bands, as well as the position of the complex between these polypeptides. Each sample contained the indicated polypeptides at 6 μM each. Samples were incubated for 10 min at room temperature to allow the formation of complexes, prior to electrophoresis at 4 °C. (C) Reconstitution of the AR activity from the ATC3 and ATN6 polypeptides using an ATP-regenerating system to remove ADP. The initial rate of GS-AMP deadenylylation was measured as described in , with GS-AMP at 8 μM, PII-UMP at 5 μM, ATP at 1 mM, α-ketoglutarate at 1 mM, KPi at 1 mM, pyruvate kinase at 0.022 unit/μL, and PEP at 5 mM. (D) Reconstitution of the AR activity from the ATC3 and ATN6 polypeptides using the noncleavable analogue AMP-PNP in place of ATP. The initial rate of GS-AMP deadenylylation was measured as described in , with GS-AMP at 16 μM, PII-UMP at 5 μM, α-ketoglutarate at 1 mM, KPi at 1 mM, and AMP-PNP at 1 mM.
Mentions: As a prelude to investigating this hypothesis, we tested whether different polypeptides derived from ATase could interact to produce the AR activity. That is, we examined whether the AR activity could be reconstituted from fragments of the ATase. Our earlier studies resulted in several polypeptides that contained various portions of the ATase (10); these were combined in all combinations (10). It was observed that in one case, a pair of polypeptides clearly associated with one another as observed by nondenaturing gel electrophoresis (10). This pair of polypeptides, called ATN6 and ATC3 (Figure 2A), when complexed together, results in the wild-type ATase missing one peptide bond, but in no case was AR activity obtained from combinations of polypeptides, including the case of the ATC3 and ATN6 pair (10). We will show here that our earlier failure to observe AR activity with this pair of polypeptides was due to the presence of ADP in the assay mixtures, and that in fact ATC3 and ATN6 polypeptides reconstitute the ATase, and their complex displays AR activity that is regulated by PII-UMP, PII, and glutamine (see ).

Bottom Line: Specifically, our results are consistent with the protein activators (PII and PII-UMP) binding to the enzyme domain with the opposing activity, with intramolecular signal transduction by direct interactions between the N-terminal AR catalytic domain and the C-terminal AT catalytic domain.Similarly, glutamine inhibition of the AR activity involved intramolecular signaling between the AT and AR domains.Finally, our results are consistent with the hypothesis that the AR activity of the N-terminal domain required activation by the opposing C-terminal (AT) domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0606, USA. pejiang@umich.edu

ABSTRACT
ATase brings about the short-term regulation of glutamine synthetase (GS) by catalyzing the adenylylation and deadenylylation of GS in response to signals of cellular nitrogen status and energy. The adenylyltransferase (AT) activity of ATase is activated by glutamine and by the unmodified form of the PII signal transduction protein and is inhibited by PII-UMP. Conversely, the adenylyl-removing (AR) activity of ATase is activated by PII-UMP and inhibited by unmodified PII and by glutamine. Here, we show that the enzyme can be reconstituted from two purified polypeptides that comprise the N-terminal two-thirds of the protein and the C-terminal one-third of the protein. Properties of the reconstituted enzyme support recent hypotheses for the sites of regulatory interactions and mechanisms for intramolecular signal transduction. Specifically, our results are consistent with the protein activators (PII and PII-UMP) binding to the enzyme domain with the opposing activity, with intramolecular signal transduction by direct interactions between the N-terminal AR catalytic domain and the C-terminal AT catalytic domain. Similarly, glutamine inhibition of the AR activity involved intramolecular signaling between the AT and AR domains. Finally, our results are consistent with the hypothesis that the AR activity of the N-terminal domain required activation by the opposing C-terminal (AT) domain.

Show MeSH
Related in: MedlinePlus