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Metabolic regulation of nitrogen fixation in Rhodospirillum rubrum
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. (Stefan Nordlund)
2009 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Nitrogen, along with carbon, hydrogen and oxygen, is amongst the most abundant elements in all living cells. The capability to convert atmospheric dinitrogen to biologically usable nitrogen compounds is only found in some prokaryotes. Biological nitrogen fixation, the reduction of dinitrogen to ammonia, is the entry step into the global nitrogen cycle. Nitrogenase, the enzyme responsible for dinitrogen reduction, requires large amounts of ATP and reducing equivalents. Consequently, the nitrogen fixation process is subjected to sophisticated regulatory networks that respond to multiple environmental stimuli. In the free-living photosynthetic nitrogen-fixing bacterium Rhodospirillum rubrum, the activity of nitrogenase is tightly regulated at the post-translational level by reversible ADP-ribosylation in response to cellular changes in nitrogen and energy status, the so-called “switch-off” effect. Our studies have been focused on identifying the intracellular signal(s) and protein components acting during “switch-off”, and elucidating the mechanism underlying this regulation. We have shown that PII signal transduction proteins and the ammonium transporter AmtB1 play central roles in the signal transduction pathway leading to the post-translational regulation of nitrogenase, in particular, the involvement of AmtB1-PII complex formation during ammonium “switch-off”. In contrast, a different signaling pathway is operating during the energy “switch-off”, and several interesting differences are highlighted here. In addition, we have solved a high-resolution structure of Dinitrogenase Reductase Activating Glycohydrolase (DRAG) using X-ray crystallography. A detailed mechanism of ADP-ribose removal by DRAG is proposed, with our structural and functional studies on DRAG supporting a reversible membrane association mechanism of regulating its activity, further controlling the activity of nitrogenase.

sted, utgiver, år, opplag, sider
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2009. , s. 64
Emneord [en]
Rhodospirillum rubrum, nitrogen fixation, switch-off, PII, AmtB1, DRAG
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
URN: urn:nbn:se:su:diva-29404ISBN: 978-91-7155-920-3 (tryckt)OAI: oai:DiVA.org:su-29404DiVA, id: diva2:232905
Disputas
2009-10-02, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Submitted.Tilgjengelig fra: 2009-09-10 Laget: 2009-08-26 Sist oppdatert: 2009-09-08bibliografisk kontrollert
Delarbeid
1. Reversible membrane association of dinitrogenase reductase activating glycohydrolase in the regulation of nitrogenase activity in Rhodospirillum rubrum; dependence on GlnJ and AmtB1
Åpne denne publikasjonen i ny fane eller vindu >>Reversible membrane association of dinitrogenase reductase activating glycohydrolase in the regulation of nitrogenase activity in Rhodospirillum rubrum; dependence on GlnJ and AmtB1
2005 (engelsk)Inngår i: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 253, nr 2, s. 273-279Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

In the photosynthetic bacterium Rhodospirillum rubrum nitrogenase activity is regulated by reversible ADP-ribosylation of dinitrogenase reductase in response to external so called “switch-off” effectors. Activation of the modified, inactive form is catalyzed by dinitrogenase reductase activating glycohydrolase (DRAG) which removes the ADP-ribose moiety. This study addresses the signal transduction between external effectors and DRAG. R. rubrum, wild-type and PII mutant strains, were studied with respect to DRAG localization. We conclude that GlnJ clearly has an effect on the association of DRAG to the membrane in agreement with the effect on regulation of nitrogenase activity. Furthermore, we have generated a R. rubrum mutant lacking the putative ammonium transporter AmtB1 which was shown not to respond to “switch-off” effectors; no loss of nitrogenase activity and no ADP-ribosylation. Interestingly, DRAG was mainly localized to the cytosol in this mutant. Overall the results support our model in which association to the membrane is part of the mechanism regulating DRAG activity.

Emneord
Nitrogenase regulation; Rhodospirillum rubrum; AmtB
Identifikatorer
urn:nbn:se:su:diva-14862 (URN)10.1016/j.femsle.2005.09.049 (DOI)16243452 (PubMedID)
Tilgjengelig fra: 2008-11-06 Laget: 2008-11-06 Sist oppdatert: 2017-12-13bibliografisk kontrollert
2. Transcription of the PII-AmtB encoding operons in Rhodospirillum rubrum and studies of the functional role(s) of GlnB, GlnJ and AmtB1 in nitrogen metabolism
Åpne denne publikasjonen i ny fane eller vindu >>Transcription of the PII-AmtB encoding operons in Rhodospirillum rubrum and studies of the functional role(s) of GlnB, GlnJ and AmtB1 in nitrogen metabolism
Vise andre…
(engelsk)Artikkel i tidsskrift (Fagfellevurdert) Submitted
Identifikatorer
urn:nbn:se:su:diva-29587 (URN)
Tilgjengelig fra: 2009-09-07 Laget: 2009-09-07 Sist oppdatert: 2009-09-08bibliografisk kontrollert
3. Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG
Åpne denne publikasjonen i ny fane eller vindu >>Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG
2009 (engelsk)Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 106, nr 34, s. 14247-14252Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

ADP-ribosylation is a ubiquitous regulatory posttranslational modification involved in numerous key processes such as DNA repair, transcription, cell differentiation, apoptosis, and the pathogenic mechanism of certain bacterial toxins. Despite the importance of this reversible process, very little is known about the structure and mechanism of the hydrolases that catalyze removal of the ADP-ribose moiety. In the phototrophic bacterium Rhodospirillum rubrum, dinitrogenase reductase-activating glycohydrolase (DraG), a dimanganese enzyme that reversibly associates with the cell membrane, is a key player in the regulation of nitrogenase activity. DraG has long served as a model protein for ADP-ribosylhydrolases. Here, we present the crystal structure of DraG in the holo and ADP-ribose bound forms. We also present the structure of a reaction intermediate analogue and propose a detailed catalytic mechanism for protein de-ADP-ribosylation involving ring opening of the substrate ribose. In addition, the particular manganese coordination in DraG suggests a rationale for the enzyme's preference for manganese over magnesium, although not requiring a redox active metal for the reaction.

Identifikatorer
urn:nbn:se:su:diva-29612 (URN)10.1073/pnas.0905906106 (DOI)000269295100017 ()
Tilgjengelig fra: 2009-09-08 Laget: 2009-09-08 Sist oppdatert: 2017-12-13bibliografisk kontrollert
4. Nitrogenase switch-off and regulation of ammonium assimilation in response to light deprivation in Rhodospirillum rubrum are influnced by the nitrogen source used during growth
Åpne denne publikasjonen i ny fane eller vindu >>Nitrogenase switch-off and regulation of ammonium assimilation in response to light deprivation in Rhodospirillum rubrum are influnced by the nitrogen source used during growth
2010 (engelsk)Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 192, nr 5, s. 1463-1466Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Nitrogen fixation and ammonium assimilation in Rhodospirillum rubrum are regulated in response to changes in light availability, and we show that the response in terms of glutamine synthetase activity and PII modification is dependent on the nitrogen source used for growth, N2 or glutamate, although both lead to nitrogenase derepression.

HSV kategori
Identifikatorer
urn:nbn:se:su:diva-29611 (URN)10.1128/JB.01456-09 (DOI)
Tilgjengelig fra: 2009-09-08 Laget: 2009-09-08 Sist oppdatert: 2017-12-13bibliografisk kontrollert

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