A novel two-domain architecture within the amino acid kinase enzyme family revealed by the crystal structure of Escherichia coli glutamate 5-kinase

Glutamate 5-kinase (G5K) makes the highly unstable product glutamyl 5-phosphate (G5P) in the initial, controlling step of proline/ornithine synthesis, being feedback-inhibited by proline or ornithine, and causing, when defective, clinical hyperammonaemia. We determined two crystal structures of G5K from Escherichia coli, at 2.9 A and 2.5 A resolution, complexed with glutamate and sulphate, or with G5P, sulphate and the proline analogue 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 characteristic alpha(3)beta(8)alpha(4) sandwich topology. This domain is responsible for catalysis and proline inhibition, and has a crater on the beta sheet C-edge that hosts the active centre and bound 5-oxoproline. Each subunit contains a 93 residue C-terminal PUA domain, typical of RNA-modifying enzymes, which presents the characteristic beta(5)beta(4) sandwich fold and three alpha 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 for 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.     

Site-directed mutagenesis studies of acetylglutamate synthase delineate the site for the arginine inhibitor

N-acetyl-l-glutamate synthase (NAGS), the first enzyme of bacterial/plant arginine biosynthesis and an essential activator of the urea cycle in animals, is, respectively, arginine-inhibited and activated. Site-directed mutagenesis of recombinant Pseudomonas aeruginosa NAGS (PaNAGS) delineates the arginine site in the PaNAGS acetylglutamate kinase-like domain, and, by extension, in human NAGS. Key residues for glutamate binding are identified in the acetyltransferase domain. However, the acetylglutamate kinase-like domain may modulate glutamate binding, since one mutation affecting this domain increases the K_m for glutamate. The effects on PaNAGS of two mutations found in human NAGS deficiency support the similarity of bacterial and human NAGSs despite their low sequence identity.     

N-Acetyl-l-Glutamate Kinase (NAGK) from Oxygenic Phototrophs: PII Signal Transduction across Domains of Life Reveals Novel Insights in NAGK Control

N-Acetyl-l-glutamate kinase (NAGK) catalyzes the first committed step in arginine biosynthesis in organisms that perform the cyclic pathway of ornithine synthesis. In eukaryotic and bacterial oxygenic phototrophs, the activity of NAGK is controlled by the P_II signal transduction protein. Recent X-ray analysis of NAGK-P_II complexes from a higher plant (Arabidopsis thaliana) and a cyanobacterium (Synechococcus elongatus) revealed that despite several differences, the overall structure of the complex is highly similar. The present study analyzes the functional conservation of P_II-mediated NAGK regulation in plants and cyanobacteria to distinguish between universal properties and those that are specific for the different phylogenetic lineages. This study shows that plant and cyanobacterial P_II proteins can mutually regulate the NAGK enzymes across the domains of life, implying a high selective pressure to conserve P_II-NAGK interaction over more than 1.2 billion years of separate evolution. The non-conserved C-terminus of S. elongatus NAGK was identified as an element, which strongly enhances arginine inhibition and is responsible for most of the differences between S. elongatus and A. thaliana NAGK with respect to arginine sensitivity. Both P_II proteins relieve arginine inhibition of NAGK, and in both lineages, P_II-mediated relief from arginine inhibition is antagonized by 2-oxoglutarate. Together, these properties highlight the conserved role of P_II as a signal integrator of the C/N balance sensed as 2-oxoglutarate to regulate arginine synthesis in oxygenic phototrophs.     

Upregulation of Dendritic Arborization by N-acetyl-D-Glucosamine Kinase Is Not Dependent on Its Kinase Activity

N-acetylglucosamine kinase (GlcNAc kinase or NAGK; EC 2.7.1.59) is highly expressed and plays a critical role in the development of dendrites in brain neurons. In this study, the authors conducted structure-function analysis to verify the previously proposed 3D model structure of GlcNAc/ATP-bound NAGK. Three point NAGK mutants with different substrate binding capacities and reaction velocities were produced. Wild-type (WT) NAGK showed strong substrate preference for GlcNAc. Conversion of Cys143, which does not make direct hydrogen bonds with GlcNAc, to Ser (i.e., C143S) had the least affect on the enzymatic activity of NAGK. Conversion of Asn36, which plays a role in domain closure by making a hydrogen bond with GlcNAc, to Ala (i.e., N36A) mildly reduced NAGK enzyme activity. Conversion of Asp107, which makes hydrogen bonds with GlcNAc and would act as a proton acceptor during nucleophilic attack on the γ-phosphate of ATP, to Ala (i.e., D107A), caused a total loss in enzyme activity. The over-expression of EGFP-tagged WT or any of the mutant NAGKs in rat hippocampal neurons (DIV 5-9) increased dendritic architectural complexity. Finally, the overexpression of the small, but not of the large, domain of NAGK resulted in dendrite degeneration. Our data show the effect of structure on the functional aspects of NAGK, and in particular, that the small domain of NAGK, and not its NAGK kinase activity, plays a critical role in the upregulation of dendritogenesis.     

A Novel Signal Transduction Protein PII Variant from Synechococcus elongatus PCC 7942 Indicates a Two-Step Process for NAGK-PII Complex Formation

P_II signal transduction proteins are highly conserved in bacteria, archaea and plants and have key functions in coordination of central metabolism by integrating signals from the carbon, nitrogen and energy status of the cell. In the cyanobacterium Synechococcus elongatus PCC 7942, P_II binds ATP and 2-oxoglutarate (2-OG) in a synergistic manner, with the ATP binding sites also accepting ADP. Depending on its effector molecule binding status, P_II (from this cyanobacterium and other oxygenic phototrophs) complexes and regulates the arginine-controlled enzyme of the cyclic ornithine pathway, N-acetyl-l-glutamate kinase (NAGK), to control arginine biosynthesis. To gain deeper insights into the process of P_II binding to NAGK, we searched for P_II variants with altered binding characteristics and found P_II variants I86N and I86T to be able to bind to an NAGK variant (R233A) that was previously shown to be unable to bind wild-type P_II protein. Analysis of interactions between these P_II variants and wild-type NAGK as well as with the NAGK R233A variant suggested that the P_II I86N variant was a superactive NAGK binder. To reveal the structural basis of this property, we solved the crystal structure of the P_II I86N variant at atomic resolution. The large T-loop, which prevails in most receptor interactions of P_II proteins, is present in a tightly bended conformation that mimics the T-loop of S. elongatus P_II after having latched onto NAGK. Moreover, both P_II I86 variants display a specific defect in 2-OG binding, implying a role of residue I86 in 2-OG binding. We propose a two-step model for the mechanism of P_II-NAGK complex formation: in an initiating step, a contact between R233 of NAGK and E85 of P_II initiates the bending of the extended T-loop of P_II, followed by a second step, where a bended T-loop deeply inserts into the NAGK clefts to form the tight complex.     

Regulation of ammonium assimilation in Haloferax mediterranei: Interaction between glutamine synthetase and two GlnK proteins

GlnK proteins belong to the PII superfamily of signal transduction proteins and are involved in the regulation of nitrogen metabolism. These proteins are normally encoded in an operon together with the structural gene for the ammonium transporter AmtB. Haloferax mediterranei possesses two genes encoding for GlnK, specifically, glnK₁ and glnK₂. The present study marks the first investigation of PII proteins in haloarchaea, and provides evidence for the direct interaction between glutamine synthetase and both GlnK₁ and GlnK₂. Complex formation between glutamine synthetase and the two GlnK proteins is demonstrated with pure recombinant protein samples using in vitro activity assays, gel filtration chromatography and western blotting. This protein–protein interaction increases glutamine synthetase activity in the presence of 2-oxoglutarate. Separate experiments that were carried out with GlnK₁ and GlnK₂ produced equivalent results.     

Two Crystal Structures of Escherichia coli N-Acetyl-l-Glutamate Kinase Demonstrate the Cycling between Open and Closed Conformations

N-Acetyl-l-glutamate kinase (NAGK), the paradigm enzyme of the amino acid kinase family, catalyzes the second step of arginine biosynthesis. Although substrate binding and catalysis were clarified by the determination of four crystal structures of the homodimeric Escherichia coli enzyme (EcNAGK), we now determine 2 Å resolution crystal structures of EcNAGK free from substrates or complexed with the product N-acetyl-l-glutamyl-5-phosphate (NAGP) and with sulfate, which reveal a novel, very open NAGK conformation to which substrates would associate and from which products would dissociate. In this conformation, the C-domain, which hosts most of the nucleotide site, rotates ∼ 24°-28° away from the N-domain, which hosts the acetylglutamate site, whereas the empty ATP site also exhibits some changes. One sulfate is found binding in the region where the β-phosphate of ATP normally binds, suggesting that ATP is first anchored to the β-phosphate site, before perfect binding by induced fit, triggering the shift to the closed conformation. In contrast, the acetylglutamate site is always well formed, although its β-hairpin lid is found here to be mobile, being closed only in the subunit of the EcNAGK-NAGP complex that binds NAGP most strongly. Lid closure appears to increase the affinity for acetylglutamate/NAGP and to stabilize the closed enzyme conformation via lid-C-domain contacts. Our finding of NAGP bound to the open conformation confirms that this product dissociates from the open enzyme form and allows reconstruction of the active center in the ternary complex with both products, delineating the final steps of the reaction, which is shown here by site-directed mutagenesis to involve centrally the invariant residue Gly11.