Unique mechanistic features of post‐translational regulation of glutamine synthetase activity in <i>Methanosarcina mazei</i> strain Gö1 in response to nitrogen availability

Ehlers, Claudia; Weidenbach, Katrin; Veit, Katharina; Forchhammer, Karl; Schmitz, Ruth A.

doi:10.1111/j.1365-2958.2005.04511.x

Cited by 56 publications

(87 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The T-loop domain, also the site of covalent modification, has been shown to be important in these interactions (17). The roles of PII proteins in regulation of nitrogen metabolism have also been studied in cyanobacteria (18) and Gram-positive bacteria (19)(20)(21), as well as in Archaea (22) and plastids (23). Although glutamine seems to be the primary nitrogen signal (reflecting excess) sensed by PII proteins in enteric bacteria (24), 2OG may be the main signal (reflecting nitrogen deficiency) in some cyanobacteria (25).…”

mentioning

confidence: 99%

Regulation of nitrogenase by 2-oxoglutarate-reversible, direct binding of a PII-like nitrogen sensor protein to dinitrogenase

Dodsworth

Leigh

2006

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Posttranslational regulation of nitrogenase, or switch-off, in the methanogenic archaeon Methanococcus maripaludis requires both nifI 1 and nifI2, which encode members of the PII family of nitrogenregulatory proteins. Previous work demonstrated that nitrogenase activity in cell extracts was inhibited in the presence of NifI 1 and NifI 2, and that 2-oxoglutarate (2OG), a potential signal of nitrogen limitation, relieved this inhibition. To further explore the role of the NifI proteins in switch-off, we found proteins that interact with NifI 1 and NifI2 and determined whether 2OG affected these interactions. Anaerobic purification of His-tagged NifI 2 resulted in copurification of NifI 1 and the dinitrogenase subunits NifD and NifK, and 2OG or a deletion mutation affecting the T-loop of NifI 2 prevented copurification of dinitrogenase but did not affect copurification of NifI 1. Similar results were obtained with His-tagged NifI 1. Gel-filtration chromatography demonstrated an interaction between purified NifI 1,2 and dinitrogenase that was inhibited by 2OG. The NifI proteins themselves formed a complex of Ϸ85 kDa, which appeared to further oligomerize in the presence of 2OG. NifI 1,2 inhibited activity of purified nitrogenase when present in a 1:1 molar ratio to dinitrogenase, and 2OG fully relieved this inhibition. These results suggest a model for switch-off of nitrogenase activity, where direct interaction of a NifI 1,2 complex with dinitrogenase causes inhibition, which is relieved by 2OG. The presence of nifI 1 and nifI2 in the nif operons of all nitrogenfixing Archaea and some anaerobic Bacteria suggests that this mode of nitrogenase regulation may operate in a wide variety of diazotrophs.ammonia switch-off ͉ Archaea ͉ Methanococcus maripaludis ͉ NifI ͉ posttranslational regulation

show abstract

mentioning

confidence: 99%

Regulation of nitrogenase by 2-oxoglutarate-reversible, direct binding of a PII-like nitrogen sensor protein to dinitrogenase

Dodsworth

Leigh

2006

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Numerous reports have documented the regulatory effects of 2OG on P II proteins targeting a variety of enzymes and transcriptional factors in other bacteria and archaea (31). Beyond P II proteins, 2OG also acts directly on other diverse proteins (14,32,51,54). Remarkably, they are all nitrogen related, implying that 2OG is a sensory metabolite that relates to cellular nitrogen status.…”

mentioning

confidence: 99%

Overcoming Fluctuation and Leakage Problems in the Quantification of Intracellular 2-Oxoglutarate Levels in Escherichia coli

Yan

Lenz

Hwa

2011

Appl Environ Microbiol

View full text Add to dashboard Cite

2-Oxoglutarate is located at the junction between central carbon and nitrogen metabolism, serving as an intermediate for both. In nitrogen metabolism, 2-oxoglutarate acts as both a carbon skeletal carrier and an effector molecule. There have been only sporadic reports of its internal concentrations. Here we describe a sensitive and accurate method for determination of the 2-oxoglutarate pool concentration in Escherichia coli. The detection was based on fluorescence derivatization followed by reversed-phase high-pressure liquid chromatography separation. Two alternative cell sampling strategies, both of which were based on a fast filtration protocol, were sequentially developed to overcome both its fast metabolism and contamination from 2-oxoglutarate that leaks into the medium. We observed rapid changes in the 2-oxoglutarate pool concentration upon sudden depletion of nutrients: decreasing upon carbon depletion and increasing upon nitrogen depletion. The latter was studied in mutants lacking either of the two enzymes using 2-oxoglutarate as the carbon substrate for glutamate biosynthesis. The results suggest that flux restriction on either reaction greatly influences the internal 2-oxoglutarate level. Additional study indicates that KgtP, a 2-oxoglutarate proton symporter, functions to recover the leakage loss of 2-oxoglutarate. This recovery mechanism benefits the measurement of cellular 2-oxoglutarate level in practice by limiting contamination from 2-oxoglutarate leakage.In enteric bacteria, 2-oxoglutarate (2OG) conjoins the two most important central metabolism pathways, being at the junction between the tricarboxylic acid (TCA) cycle and central nitrogen metabolism. Unlike central carbon metabolism, central nitrogen metabolism is rather compact and consists of only three enzymes (42), glutamine synthetase (GS; encoded by glnA), glutamate synthase (GOGAT; encoded by gltBD), and glutamate dehydrogenase (GDH; encoded by gdhA), and three metabolites, glutamine (Gln), glutamate (Glu), and 2OG (see Fig. S1 in the supplemental material). NH 4 ϩ , the preferred nitrogen source for cell growth, is assimilated through GS and GDH. 2OG from the TCA cycle serves as the sole carbon skeletal substrate, generating Glu through GOGAT and/or GDH and subsequently Gln through GS. Most of the total carbon that 2OG brings into nitrogen metabolism is recycled from the two central nitrogen intermediates: 2OG is regenerated after many transamination reactions from Glu, and Glu is regenerated after various amido transfer reactions from Gln. Almost all other cellular nitrogen is gained from these two types of nitrogen transfer reactions. Hence, 2OG has a dual identity: a central carbon intermediate and a nitrogen carrier.2OG also functions as a regulatory effector. The most notable receptors of 2OG are P II family proteins (31, 38). There are two P II proteins in Escherichia coli, GlnB and its paralog, GlnK (6, 53). GlnB is one key player in nitrogen regulation, participating in two branched regulatory cascades that control the activi...

show abstract

“…These include the transcriptional activators NifA of Gammaproteobacteria (17) and NtcA of unicellular cyanobacteria (7), the glutamine synthetase modifying adenylyltransferase of Gammaproteobacteria (9), and glutamine synthetase itself in the archaeal genus Methanosarcina (4). A recently characterized 2OG-responsive regulator that operates at the transcriptional level in a wide range of Euryarchaeota is the repressor NrpR (13).…”

mentioning

confidence: 99%

Genetic Screen for Regulatory Mutations in Methanococcus maripaludis and Its Use in Identification of Induction-Deficient Mutants of the Euryarchaeal Repressor NrpR

Lie

Leigh

2007

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

Unique mechanistic features of post‐translational regulation of glutamine synthetase activity in Methanosarcina mazei strain Gö1 in response to nitrogen availability

Cited by 56 publications

References 60 publications

Regulation of nitrogenase by 2-oxoglutarate-reversible, direct binding of a PII-like nitrogen sensor protein to dinitrogenase

Regulation of nitrogenase by 2-oxoglutarate-reversible, direct binding of a PII-like nitrogen sensor protein to dinitrogenase

Overcoming Fluctuation and Leakage Problems in the Quantification of Intracellular 2-Oxoglutarate Levels in Escherichia coli

Genetic Screen for Regulatory Mutations in Methanococcus maripaludis and Its Use in Identification of Induction-Deficient Mutants of the Euryarchaeal Repressor NrpR

Contact Info

Product

Resources

About