Basis of Arginine Sensitivity of Microbial N -Acetyl- l -Glutamate Kinases: Mutagenesis and Protein Engineering Study with the Pseudomonas aeruginosa and Escherichia coli Enzymes

Abstract

N-Acetylglutamate kinase (NAGK) catalyzes the second step of arginine biosynthesis. In Pseudomonas aeruginosa, but not in Escherichia coli, this step is rate limiting and feedback and sigmoidally inhibited by arginine. Crystal structures revealed that arginine-insensitive E. coli NAGK (EcNAGK) is homodimeric, whereas arginine-inhibitable NAGKs, including P. aeruginosa NAGK (PaNAGK), are hexamers in which an extra N-terminal kinked helix (N-helix) interlinks three dimers. By introducing single amino acid replacements in PaNAGK, we prove the functionality of the structurally identified arginine site, as arginine site mutations selectively decreased the apparent affinity for arginine. N-helix mutations affecting R24 and E17 increased and decreased, respectively, the apparent affinity of PaNAGK for arginine, as predicted from enzyme structures that revealed the respective formation by these residues of bonds favoring inaccessible and accessible arginine site conformations. N-helix N-terminal deletions spanning >16 residues dissociated PaNAGK to active dimers, those of <20 residues decreased the apparent affinity for arginine, and complete N-helix deletion (26 residues) abolished arginine inhibition. Upon attachment of the PaNAGK N-terminal extension to the EcNAGK N terminus, EcNAGK remained dimeric and arginine insensitive. We concluded that the N-helix and its C-terminal portion after the kink are essential but not sufficient for hexamer formation and arginine inhibition, respectively; that the N-helix modulates NAGK affinity for arginine and mediates signal transmission between arginine sites, thus establishing sigmoidal arginine inhibition kinetics; that the mobile ␣H-␤16 loop of the arginine site is the modulatory signal receiver; and that the hexameric architecture is not essential for arginine inhibition but is functionally essential for physiologically relevant arginine control of NAGK.Microorganisms and plants make arginine from glutamate in eight steps (6), among which the first five (glutamate3N-acetylglutamate3N-acetylglutamyl-␥-phosphate3N-acetylglutamate-␥-semialdehyde3N-acetylornithine3ornithine) involve N-acetylated intermediates and generate the arginine precursor ornithine (5,21,22). The absence of steps 2 to 5 in animals (2, 14) renders the enzymes catalyzing these steps potential candidates for developing antibacterials and biocides. In some microorganisms, such as Escherichia coli, the arginine biosynthetic route is linear because ornithine is produced by the hydrolysis of N-acetylornithine (5). In such cases, the first enzyme of the route, N-acetylglutamate synthase (NAGS), catalyzes the controlling step and is feedback inhibited by the final product, arginine. Many more organisms, including yeasts, algae, plants, and many bacteria, such as Pseudomonas aeruginosa, have a more evolved, cyclic variant of this route in which the acetyl group is recycled by its transfer from acetylornithine to glutamate (5,6,22). In this case, NAGS plays a purely anaplerotic role, and the main controlling step that ...

Pyrroline‐5‐carboxylate synthase and proline biosynthesis: From osmotolerance to rare metabolic disease

“…In principle, the oligomeric state of these enzymes could modulate the mechanisms that regulate their activities, as described for another member of the AAK family, the N ‐acetylglutamate kinase (NAGK). While NAGK from Pseudomonas aeruginosa is hexameric and inhibited by arginine, this enzyme from E. coli is dimeric and insensitive to arginine 26. However, a triple mutant of E. coli GK that can form only dimers displays the same kinetic and allosteric properties as the wild‐type enzyme, suggesting that the mechanism of allosteric inhibition in GK is independent of the oligomeric state of the enzyme (unpublished results from our laboratory).…”

N-acetylglutamate synthase: structure, function and defects

“…The amino acid sequences for the L-arginine binding loop (residue 270–280) is highly conserved across all known NAGS proteins. This universality of the L-arginine binding site is supported by the mutagenesis studies of this region in mouse NAGS and bifunctional X. campestris NAGS/K [13] and Pseudomonas aeruginosa NAGS [21] and NAGK [22]. …”

On the Conservation of the Slow Conformational Dynamics within the Amino Acid Kinase Family: NAGK the Paradigm

“…In many organisms, NAGK phosphorylation is the controlling step in arginine biosynthesis. In these cases, NAGK is feedback inhibited by the end product arginine, and recent studies shed light on this mechanism of inhibition [34] , [35] . NAGK from Escherichia Coli ( Ec NAGK), on the other hand, is arginine-insensitive.…”