Photosynthetic organisms can store nitrogen by synthesizing arginine, and, therefore, feedback inhibition of arginine synthesis must be relieved in these organisms when nitrogen is abundant. This relief is accomplished by the binding of the P II signal transduction protein to acetylglutamate kinase (NAGK), the controlling enzyme of arginine synthesis. Here, we describe the crystal structure of the complex between NAGK and P II of Synechococcus elongatus, at 2.75-Å resolution. We prove the physiological relevance of the observed interactions by site-directed mutagenesis and functional studies. The complex consists of two polar P II trimers sandwiching one ring-like hexameric NAGK (a trimer of dimers) with the threefold axes of these molecules aligned. The binding of P II favors a narrow ring conformation of the NAGK hexamer that is associated with arginine sites having low affinity for this inhibitor. Each PII subunit contacts one NAGK subunit only. The contacts map in the inner circumference of the NAGK ring and involve two surfaces of the P II subunit. One surface is on the PII body and interacts with the C-domain of the NAGK subunit, helping widen the arginine site found on the other side of this domain. The other surface is at the distal region of a protruding large loop (T-loop) that presents a novel compact shape. This loop is inserted in the interdomain crevice of the NAGK subunit, contacting mainly the N-domain, and playing key roles in anchoring P II on NAGK, in activating NAGK, and in complex formation regulation by MgATP, ADP, 2-oxoglutarate, and by phosphorylation of serine-49.arginine synthesis ͉ regulation ͉ x-ray structure ͉ signaling ͉ cyanobacteria I n photosynthetic organisms nitrogen can be stored by synthesizing arginine (1, 2) and, therefore, feedback inhibition of arginine synthesis must be relieved when nitrogen is abundant. The enzyme of arginine biosynthesis that is the target of arginine inhibition, N-acetyl-L-glutamate (NAG) kinase (NAGK) (1, 3-5), was found in cyanobacteria and plants (2, 4-8) to be a target of the carbon/ nitrogen P II signaling protein (9, 10), forming with it a complex in which arginine inhibition is alleviated (6, 7). P II signaling proteins are homotrimers of a 12-to 13-kDa subunit that interact with enzymes, transcription factors, and ammonia channels, regulating their activity (9, 10) and carbon/ nitrogen homeostasis. Numerous structures of P II proteins, including those for cyanobacteria and plants (9-12), are known, but it was unclear how P II proteins carry out their functions. The body of the P II trimer is roughly hemispheric. Its subunits have ␤␣␤␤␣␤ topology, with ␣ helices looking outward and the ␤ sheet inward and providing the intersubunit interactions. Each subunit has three loops: the B-and C-loops and the larger flexible T-loop. The T-loop residues Y51 and S49 are, respectively, the sites of the regulatory uridylylation and phosphorylation in enterobacterial and cyanobacterial P II proteins (9, 10), with S49 phosphorylation abolishing interaction wi...

PII, a homotrimeric very ancient and highly widespread (bacteria, archaea, plants) key sensor-transducer protein, conveys signals of abundance or poorness of carbon, energy and usable nitrogen, converting these signals into changes in the activities of channels, enzymes, or of gene expression. PII sensing is mediated by the PII allosteric effectors ATP, ADP (and, in some organisms, AMP), 2-oxoglutarate (2OG; it reflects carbon abundance and nitrogen scarcity) and, in many plants, L-glutamine. Cyanobacteria have been crucial for clarification of the structural bases of PII function and regulation. They are the subject of this review because the information gathered on them provides an overall structure-based view of a PII regulatory network. Studies on these organisms yielded a first structure of a PII complex with an enzyme, (N-acetyl-Lglutamate kinase, NAGK), deciphering how PII can cause enzyme activation, and how it promotes nitrogen stockpiling as arginine in cyanobacteria and plants. They have also revealed the first clear-cut mechanism by which PII can control gene expression. A small adaptor protein, PipX, is sequestered by PII when nitrogen is abundant and is released when is scarce, swapping partner by binding to the 2OG-activated transcriptional regulator NtcA, co-activating it. The structures of PII-NAGK, PII-PipX, PipX alone, of NtcA in inactive and 2OG-activated forms and as NtcA-2OG-PipX complex, explain structurally PII regulatory functions and reveal the changing shapes and interactions of the T-loops of PII depending on the partner and on the allosteric effectors bound to PII. Cyanobacterial studies have also revealed that in the PII-PipX complex PipX binds an additional transcriptional factor, PlmA, thus possibly expanding PipX roles beyond NtcA-dependency. Further exploration of these roles has revealed a functional interaction of PipX with PipY, a pyridoxal-phosphate (PLP) protein involved in PLP homeostasis whose mutations in the human ortholog cause epilepsy. Knowledge of cellular levels of the different components of this PII-PipX regulatory network and of KD values for some of the complexes provides the basic background for gross modeling of the system at high and low nitrogen abundance. The cyanobacterial network can guide searches for analogous components in other organisms, particularly of PipX functional analogs.

Summary. Glutamate 5-kinase (G5K) makes the highly unstable product glutamyl-5-phosphate (G5P) in the initial, controlling step of proline/ornithine synthesis, being feed-back inhibited by proline or ornithine, and causing, when defective, clinical hyperammonaemia. We have determined two crystal structures of G5K from Escherichia coli, at 2.9-and 2.5-Å-resolution, complexed with glutamate and sulphate, or with G5P, sulphate and the proline analog 5-oxoproline. E. coli G5K presents a novel tetrameric (dimer of dimers) architecture. Each subunit contains a 257-residue AAK domain, typical of acylphosphate-forming enzymes, with the characteristic  3  8  4 sandwich topology. This domain is responsible for catalysis and proline inhibition and has a crater on the  sheet C-edge that hosts the active centre and the bound 5-oxoproline. Each subunit also contains a 93-residue C-terminal PUA domain, typical of RNA-modifying enzymes, which presents the characteristic  5  4 sandwich fold and three  helices. The AAK and PUA domains of one subunit associate non-canonically in the dimer with the same domains of the other subunit, leaving a negatively charged hole between them that hosts two Mg ions in one crystal, in line with the G5K requirement of free Mg. The tetramer, formed by two dimers interacting exclusively through their AAK domains, is flat and elongated, and has in each face, pericentrically, two exposed active centres in alternate subunits. This would permit the close apposition of two active centres of bacterial glutamate-5-phosphate reductase (the next enzyme in the proline/ornithine-synthesising route), supporting the postulated channelling of G5P.The structures clarify substrate binding and catalysis, justify the high glutamate specificity, explain the effects of known point mutations, and support the binding of proline near glutamate. Proline binding may trigger the movement of a loop that encircles glutamate, and which participates in a hydrogen bond network connecting active centres which is possibly involved in the cooperativity for glutamate.

No abstract

Δ(1)-Pyrroline-5-carboxylate synthetase (P5CS) catalyzes the first two steps of ornithine/proline biosynthesis. P5CS deficiency has been reported in three families, with patients presenting with cutis/joint laxity, cataracts, and neurodevelopmental delay. Only one family exhibited metabolic changes consistent with P5CS deficiency (low proline/ornithine/citrulline/arginine; fasting hyperammonemia). Here we report a new P5CS-deficient patient presenting the complete clinical/metabolic phenotype and carrying p.G93R and p.T299I substitutions in the γ-glutamyl kinase (γGK) component of P5CS. The effects of these substitutions are (1) tested in mutagenesis/functional studies with E.coli γGK, (2) rationalized by structural modelling, and (3) reflected in decreased P5CS protein in patient fibroblasts (shown by immunofluorescence). Using optical/electron microscopy on skin biopsy, we show collagen/elastin fiber alterations that may contribute to connective tissue laxity and are compatible with our angio-MRI finding of kinky brain vessels in the patient. MR spectroscopy revealed decreased brain creatine, which normalized after sustained arginine supplementation, with improvement of neurodevelopmental and metabolic parameters, suggesting a pathogenic role of brain creatine decrease and the value of arginine therapy. Morphological and functional studies of fibroblast mitochondria show that P5CS deficiency is not associated with the mitochondrial alterations observed in Δ(1)-pyrroline-5-carboxylate reductase deficiency (another proline biosynthesis defect presenting cutis laxa and neurological alterations).

No abstract

No abstract

No abstract