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The activity of adenylyltransferase in Rhodospirillum rubrum is only affected by alpha-ketoglutarate and unmodified PII proteins, but not by glutamine, in vitro
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2007 (English)In: The FBS Journal, ISSN 1742-464X, Vol. 274, no 10, 2449-2460 p.Article in journal (Refereed) Published
Abstract [en]

Ammonium assimilation is tightly regulated in nitrogen-fixing bacteria; the target of regulation is primarily the activity of the key enzyme glutamine synthetase that is regulated by reversible covalent modification by AMP groups in reactions catalysed by the bifunctional adenylyltransferase (ATase). The properties and regulation of ATase from Escherichia coli have been studied in great detail. We have investigated the regulation of ATase from Rhodospirillum rubrum, a photosynthetic nitrogen-fixing bacterium. In this diazotroph, nitrogenase is regulated at the metabolic level in addition to the transcriptional regulation operating in all diazotrophic bacteria, which makes understanding the regulatory features of nitrogen assimilation even more interesting. We show that in R. rubrum, in contrast to the E. coli system, ATase is primarily regulated by α-ketoglutarate and that glutamine has no effect on neither the adenylylation nor the deadenylylation of glutamine synthetase. Furthermore, the role of the regulatory PII proteins is only to stimulate the adenylylation reaction, as there is no effect on the reverse reaction. We propose that in R. rubrum and possibly other diazotrophs α-ketoglutarate plays the central role in the regulation of ATase and thus glutamine synthetase activity.

Place, publisher, year, edition, pages
2007. Vol. 274, no 10, 2449-2460 p.
Keyword [en]
adenylyltransferase, ammonium assimilation, glutamine synthetase, Rhodospirillum rubrum
Identifiers
URN: urn:nbn:se:su:diva-10353DOI: 10.1111/j.1742-4658.2007.05778.xISI: 000246029800002PubMedID: 17419734OAI: oai:DiVA.org:su-10353DiVA: diva2:176872
Available from: 2007-12-28 Created: 2007-12-28 Last updated: 2010-01-14Bibliographically approved
In thesis
1. PII proteins as global regulators of bacterial nitrogen metabolism
Open this publication in new window or tab >>PII proteins as global regulators of bacterial nitrogen metabolism
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Nitrogen is an essential element to sustain life, being a component of most biological macromolecules. In spite of the abundance of gaseous N2, the availability of nitrogen compounds that can be readily used by most microorganisms is scarce and its production energetically demanding. Due to the central importance of nitrogen metabolism, most microorganisms evolved elaborate mechanisms to ensure efficient regulation, balancing substrate availability, product formation and energy expenditure.

In most bacteria, many archaea and some plants, the different aspects of nitrogen metabolism are coordinated by members of the PII family of signal transduction proteins, acting as fundamental molecular messengers controlling several cellular processes. In proteobacteria, including the nitrogen fixing organism Rhodospirillum rubrum, these proteins are involved in regulation at different levels: they regulate gene expression, modulating the activity of several transcription factors; they control the flux through the ammonium transport protein (AmtB); they influence the activity of key metabolic enzymes, e.g. glutamine synthetase (GS) and nitrogenase. The signal sensing and integration by these proteins is achieved in two different yet interdependent strategies: allosteric regulation (by the binding of metabolites like ATP, ADP, 2-oxoglutarate) and reversible post-translational modification. Signal integration likely results in different conformations of the proteins, influencing the direct protein-protein interaction with the cellular targets.

In the present work, using R. rubrum as a model organism, we have studied some aspects of the biochemistry of PII proteins in terms of regulatory interactions with the ammonium transport protein AmtB1 and the adenylyltransferase GlnE (involved in GS regulation). Additionally, we have investigated the post-translational modification of PII proteins, showing for the first time in vivo in addition in vitro selectivity in the modification of different PII proteins.

Our results contributed to elucidate several new aspects in the regulation by PII proteins and also strengthened the idea that these proteins act as global regulators in the context of bacterial nitrogen metabolism.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholms universitet, 2009. 68 p.
Keyword
Rhodospirillum rubrum, nitrogen metabolism, signal transduction, PII proteins
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-30815 (URN)978-91-7155-963-0 (ISBN)
Public defence
2009-12-07, Magnélisalen, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 1: Manuscript. Paper 4: Manuscript.Available from: 2009-11-15 Created: 2009-10-27 Last updated: 2010-01-12Bibliographically approved
2. Regulation of Glutamine Synthetase in the Diazotroph Rhodospirillum rubrum
Open this publication in new window or tab >>Regulation of Glutamine Synthetase in the Diazotroph Rhodospirillum rubrum
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The bacterial cell needs ammonia for synthesis of glutamine from glutamate. Only one enzyme is able to catalyze this reaction, namely glutamine synthetase (GS). GS can be regulated both transcriptionally and post-translationally and it is present in all kingdoms of life. Our study has been focused on the post-translational regulation of GS in the diazotrophic bacterium Rhodospirillum rubrum. A number of proteins are involved in the covalent regulation of GS, among them are the regulatory PII proteins that depending on growth conditions also like GS are covalently modified. We have purified all proteins involved in GS regulation and developed several in vitro assays with the aim of understanding GS regulation in R. rubrum. Studies on the influence of the small metabolite effectors α-ketoglutarate and glutamine are also included together with the effect of divalent cations.

In both R. rubrum and Escherichia coli, one of the enzymes participating in GS regulation is the bifunctional enzyme GlnE. GlnE is responsible for both the attachment and the removal of AMP groups from GS, which basically leads to a more inactive or active enzyme respectively. Apart from examining the above functions of GlnE, we have also found a novel third activity of R. rubrum GlnE, an antioxidant function, which is located in the C-terminal domain. We have examined this novel activity of GlnE in great detail, including site specific mutagenesis.

We also generated and analyzed ΔglnE mutants in R. rubrum and the results from these studies show that suppressor mutations can occur within glnA, the gene encoding GS. We assume that the function of these suppressor mutations is to lower the specific activity of GS, which otherwise might be too high in a ΔglnE mutant since they lack the ability to adenylylate GS. In other words, it seems that ΔglnE mutants can not be generated without producing suppressor mutations.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2007. 63 p.
Keyword
glutamine synthetase, Rhodospirillum rubrum, GlnE, PII
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-7050 (URN)978-91-7155-495-6 (ISBN)
Public defence
2007-10-01, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 14:00 (English)
Opponent
Supervisors
Available from: 2007-09-10 Created: 2007-09-03 Last updated: 2010-01-12Bibliographically approved

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