g glutamine synthetase (GS) and nitrogenase [5, 6] PII proteins

g. glutamine synthetase (GS) and nitrogenase [5, 6]. PII proteins are trimers of about 37 kDa, with each monomer containing a double βαβ ferredoxin fold. It GDC-0941 concentration has been previously shown that each trimer

can bind up to three molecules of 2-oxoglutarate (2-OG) and ATP/ADP allowing the sensing of the carbon/nitrogen and energy status in the cell [7, 8]. In the different structures of PII proteins solved so far, one of the most striking characteristics is the existence of three surface exposed loops per monomer, the B, C and T-loops [2]. The three nucleotide-binding sites (where ATP and ADP bind) are located in the inter-subunit clefts formed by the interaction of the B and C loops. The binding of ATP displays negative cooperativity (as does 2-OG binding), with ADP competing for the same binding site, as was shown for GlnB from Escherichia coli [7]. Recent structures of Synechococcos elongatus GlnB and Azospirillum brasilense GlnZ have convincingly elucidated the 2-OG binding sites within PII proteins

and established that this binding influences protein conformation, particularly of the T-loop region [9, 10]. Moreover, the structure of S. elongatus GlnB also provided an explanation for the negative cooperativity observed in the binding of 2-OG, considering that binding of the first 2-OG molecule generates unequal binding sites in the other two subunits [9]. In most proteobacteria, including the photosynthetic nitrogen-fixing bacterium Rhodospirillum BIBW2992 rubrum, PII proteins are covalently modified by reversible uridylylation at tyrosine 51 in the T-loop, yielding 0–3 subunits modified with UMP per trimer. The uridylyltransferase and uridylylremoving activities are catalyzed by the bifunctional enzyme uridylyltransferase GlnD, with the reactions

being regulated Thymidylate synthase by the concentration of 2-oxoglutarate, through binding to the PII proteins [11]. The two activities of R. rubrum GlnD occur at distinct active sites, with the N-terminal nucleotidyltransferase domain involved in PII uridylylation and the central HD domain responsible for PII-UMP deuridylylation [12]. In R. rubrum, three PII proteins have been identified and named GlnB, GlnJ and GlnK [6]. However, only GlnB and GlnJ have been extensively studied and found to have both unique and overlapping functions in the regulation of gene transcription (two-component system NtrBC), ammonium transport (AmtB) and activity of metabolic Ralimetinib enzymes GS and nitrogenase (by regulating the DRAT/DRAG system). While both proteins can regulate the activity of the adenylyltransferase GlnE (and thereby controling GS activity), GlnB specifically regulates NtrB and DRAT and GlnJ has a preferential role in the regulation of AmtB and possibly DRAG [5, 6, 13–15].

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