Biochemical properties of hNAT.

<p>A and B, dependence of enzyme activity on the concentration of AcCoA or L-glutamate. AcCoA or L-glutamate was varied in the range of 0.25–5.0 and 0.50–20.0, respectively, with L-glutamate and AcCoA fixed at 10 or 2.5 mM, respectively.</p

RMSD values (Å) among different subunit within NAG bound structure and with the bifunctional mmNAGS/K native structure.

<p>RMSD values (Å) among different subunit within NAG bound structure and with the bifunctional mmNAGS/K native structure.</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

Enzyme activities of hNAT and active site mutants.

<p>Enzyme activities of hNAT and active site mutants.</p

Structure of hNAT.

<p>A: Ribbon diagram of hNAT subunit structure. Bound NAG is shown as sky-blue sticks. The electron density map (2F_o–F_c) around bound NAG (contoured at 1.0 σ) is shown as blue cage. B: Superimposition of four hNAT subunits in asymmetric unit. The bound NAG is shown as sky-blue sticks. The proposed bound CoA is shown as green sticks. Subunits A, B, X and Y are shown in pink, yellow, green and blue ribbons, respectively. C: The hNAT molecular dimer. Subunits A and B are shown in green and red ribbons, respectively. D: Details of the interactions between subunits A and B. Side-chains of the residues in the interface are shown in sticks. Potential hydrogen bonding interactions are shown in red dashed lines.</p

Superimposition of hNAT with the NAT domain of subunit X of mmNAGS/K.

<p>The structure of hNAT is shown in pink ribbons. The structure of the NAT domain of subunit X of mmNAGS/K is shown in yellow ribbons. The bound NAG is shown in sky-blue sticks. The proposed bound CoA is shown in green sticks. Residues that are mentioned in text are shown in sticks.</p

Crystal Structure of the <i>N</i>-Acetyltransferase Domain of Human <i>N</i>-Acetyl-L-Glutamate Synthase in Complex with <i>N</i>-Acetyl-L-Glutamate Provides Insights into Its Catalytic and Regulatory Mechanisms

<div><p><i>N</i>-acetylglutamate synthase (NAGS) catalyzes the conversion of AcCoA and L-glutamate to CoA and <i>N</i>-acetyl-L-glutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic <i>N</i>-acetyltransferase (hNAT) domain with <i>N</i>-acetyl-L-glutamate bound at 2.1 Å resolution. Functional studies indicate that the hNAT domain retains catalytic activity in the absence of the amino acid kinase (AAK) domain. Instead, the major functions of the AAK domain appear to be providing a binding site for the allosteric activator, L-arginine, and an <i>N</i>-terminal proline-rich motif that is likely to function in signal transduction to CPS1. Crystalline hNAT forms a dimer similar to the NAT-NAT dimers that form in crystals of bifunctional <i>N</i>-acetylglutamate synthase/kinase (NAGS/K) from <i>Maricaulis maris</i> and also exists as a dimer in solution. The structure of the NAG binding site, in combination with mutagenesis studies, provide insights into the catalytic mechanism. We also show that native NAGS from human and mouse exists in tetrameric form, similar to those of bifunctional NAGS/K.</p></div

Oligomeric structure of mNAGS and hNAGS in solution.

<p>A: Analytic gel chromatography of mNAGS and hNAGS. Elution profiles of mNAGS and hNAGS are shown in dashed and solid lines, respectively. B: Analytic gel chromatography of mNAT and hNAT. Elution profiles of mNAT and hNAT are shown in dashed and solid lines, respectively. C: Cross-linking of mNAGS. Lanes 1; protein size markers; 2, mNAGS (2.5 µg) without cross-linking reagent; 3, mNAGS (2.5 µg) with cross-linking reagent, suberic acid bis(3-sulfo-N-hydroxysuccinimide ester) sodium salt; 4, mNAGS with cross-linking reagent, dimethyl suberimidate dihydrochloride. D: Cross-linking of mNAGS. Lanes 1; protein size markers; 2, mNAGS without cross-linking reagent; 3, mNAGS (1.5 µg) with cross-linking reagent, dimethyl suberimidate dihydrochloride; 4, mNAGS (4.5 µg) with cross-linking reagent, dimethyl suberimidate dihydrochloride.</p

Stereo diagram of the proposed CoA binding site.

<p>The proposed bound CoA is shown in green sticks. The bound NAG is shown in sky-blue sticks. Side-chains of residues that potentially hydrogen bond to CoA are shown in yellow sticks. The water molecule (w37) that occupies the similar position of thiol S of CoA is shown in a red ball.</p

NAG binding site.

<p>A: Stereo diagram of NAG binding site. The bound NAG is shown in sky-blue sticks. The side-chains involved in hydrogen bonding interactions with NAG are shown in green sticks. The side-chains of other surrounding residues are shown in yellow sticks. The water molecule (w37) is shown in red ball. The electron density map (2F_o–F_c) around bound NAG (contoured at 1.0 σ) is shown as blue cage. Potential hydrogen bonding interactions are shown in red dashed lines. B: Stereo diagram of “water wire” channel. The bound NAG is shown in sky-blue sticks. Water molecules are shown in yellow balls. Residues involved in hydrogen bonding interactions are shown in brown sticks. Potential hydrogen bonding interactions are shown in red dashed lines.</p