N-acetylglutamate synthase: structure, function and defects

Abstract

N-acetylglutamate (NAG) is a unique enzyme cofactor, essential for liver ureagenesis in mammals while it is the first committed substrate for de novo arginine biosynthesis in microorganisms and plants. The enzyme that produces NAG from glutamate and CoA, NAG synthase (NAGS), is allosterically inhibited by arginine in microorganisms and plants and activated in mammals. This transition of the allosteric effect occurred when tetrapods moved from sea to land. The first mammalian NAGS gene (from mouse) was cloned in 2002 and revealed significant differences from the NAGS ortholog in microorganisms. Almost all NAGS genes possess a C-terminus transferase domain in which the catalytic activity resides and an N-terminus kinase domain where arginine binds. The three-dimensional structure of NAGS shows two distinctly folded domains. The kinase domain binds arginine while the acetyltransferase domain contains the catalytic site. NAGS deficiency in humans leads to hyperammonemia and can be primary, due to mutations in the NAGS gene or secondary due to other mitochondrial aberrations that interfere with the normal function of the same enzyme. For either condition, N-carbamylglutamate (NCG), a stable functional analog of NAG, was found to either restore or improve the deficient urea-cycle function.

Figure 1

Conservation of mammalian NAGS. Logo representation of the conservation of amino acids among NAGS from human, mouse, rat, dog, horse and cow. The letter size is proportional to the degree of conservation and the color indicates the type of amino acid. Post-translational processing sites found upon expression of mouse NAGS in insect cells are indicated by arrows. MTS - mitochondrial targeting sequence.

Figure 2

Structure of N. gonorrhoeae NAGS. Ribbon diagram of a monomer (A), and its AAK (B) and NAT (C) domains. Green arrows indicate the direction of strands in β-sheets, α-helices are in red and β-sheets are in green. AcCoA is represented as a stick model.

Figure 3

N. gonorrhoeae NAGS L-arginine binding site in the T-state structure. A. Ribbon diagram of T-state monomer. Ribbons are shown in rainbow colors from blue (N-terminal) to red (C-terminal). Bound L-arginine is represented as space-filling models. Bound CoA is shown as green sticks. B. Arginine binding site. Electron density maps (2F_o-F_c) are shown as a blue cage contoured at 1.0σ. Carbon atoms of L-arginine are shown as pink sticks. Carbon atoms of the side chains to interacts with L-arginine directly and indirectly are shown as yellow and green sticks, respectively. Hydrogen bonds between bound ligands and protein are indicated by red dashed lines.

Figure 4

Molecular hexamer of N. gonorrhoeae NAGS. Simplified model showing the quaternary interactions of the subunits. Two dimer interfaces across the two-fold axes between AAK domain K1 and K4, and K1 and K5, and one interface across the three-fold axis between the NAT domain S1 and the AAK domain K2 are clearly visible. Different subunits are shown in different colors.

Figure 5

Increase over time of isotopic enrichment in plasma [¹³C] urea (A) and the concentration of plasma [¹³C] urea (B) in a patient with NAGS deficiency before (○) and after (●) 3-d treatment with N-carbamylglutamate.