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Regulation of Glutamine Synthetase in the Diazotroph Rhodospirillum rubrum
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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 [en]
glutamine synthetase, Rhodospirillum rubrum, GlnE, PII
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-7050ISBN: 978-91-7155-495-6 (print)OAI: oai:DiVA.org:su-7050DiVA: diva2:197555
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
List of papers
1. In vitro studies of the uridylylation of the three PII protein paralogs from Rhodospirillum rubrum is only affected by alfa-ketoglutarate and divalent cations but not by glutamine
Open this publication in new window or tab >>In vitro studies of the uridylylation of the three PII protein paralogs from Rhodospirillum rubrum is only affected by alfa-ketoglutarate and divalent cations but not by glutamine
2007 In: Journal of Bacteriology, ISSN 0021-9193, Vol. 189, 3471-3478 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-24451 (URN)000246028400015 ()
Note
Part of urn:nbn:se:su:diva-7050Available from: 2007-09-10 Created: 2007-09-03Bibliographically approved
2. The activity of adenylyltransferase in Rhodospirillum rubrum is only affected by alpha-ketoglutarate and unmodified PII proteins, but not by glutamine, in vitro
Open this publication in new window or tab >>The activity of adenylyltransferase in Rhodospirillum rubrum is only affected by alpha-ketoglutarate and unmodified PII proteins, but not by glutamine, in vitro
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.

Keyword
adenylyltransferase, ammonium assimilation, glutamine synthetase, Rhodospirillum rubrum
Identifiers
urn:nbn:se:su:diva-10353 (URN)10.1111/j.1742-4658.2007.05778.x (DOI)000246029800002 ()17419734 (PubMedID)
Available from: 2007-12-28 Created: 2007-12-28 Last updated: 2010-01-14Bibliographically approved
3. A novel peroxiredoxin activity is located within the C-terminal end of Rhodospirillum rubrum adenylyltransferase.
Open this publication in new window or tab >>A novel peroxiredoxin activity is located within the C-terminal end of Rhodospirillum rubrum adenylyltransferase.
2008 (English)In: Journal of Bacteriology, ISSN 0021-9193, Vol. 190, no 1, 434-7 p.Article in journal (Refereed) Published
Abstract [en]

Adenylyltransferase (GlnE) catalyzes the reversible adenylylation of glutamine synthetase. In this report we present, for the first time, evidence for a peroxiredoxin activity of Rhodospirillum rubrum GlnE, through the carboxyl-terminal AhpC/thiol-specific antioxidant (TSA) domain. The combination of GlnE and AhpC/TSA domains within the same polypeptide constitutes a unique domain architecture that has not previously been identified among proteobacteria.

Identifiers
urn:nbn:se:su:diva-30808 (URN)10.1128/JB.01058-07 (DOI)000252080400041 ()17951375 (PubMedID)
Available from: 2009-10-27 Created: 2009-10-27 Last updated: 2010-01-13Bibliographically approved
4. Reduced activity of glutamine synthetase in Rhodospirillum rubrum mutants lacking adenylyltransferase, GlnE
Open this publication in new window or tab >>Reduced activity of glutamine synthetase in Rhodospirillum rubrum mutants lacking adenylyltransferase, GlnE
Manuscript (Other academic)
Identifiers
urn:nbn:se:su:diva-24454 (URN)
Note
Part of urn:nbn:se:su:diva-7050Available from: 2007-09-10 Created: 2007-09-03 Last updated: 2010-01-13Bibliographically approved

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