Mammalian N-acetylglutamate synthase

Abstract

N-Acetylglutamate synthase (NAGS, E.C. 2.3.1.1) is a mitochondrial enzyme that catalyzes the formation of N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI). The mouse and human NAGS genes have been identified based on similarity to regions of NAGS from Neurospora crassa and cloned from liver cDNA libraries. These genes were shown to complement an argA- (NAGS) deficient Escherichia coli strain, and enzymatic activity of the proteins was confirmed by a new stable isotope dilution assay. The deduced amino acid sequence of mammalian NAGS contains a putative mitochondrial-targeting signal at the N-terminus. The mouse NAGS preprotein was overexpressed in insect cells to determine post-translational modifications and two processed proteins with different N-terminal truncations have been identified. Sequence analysis using a hidden Markov model suggests that the vertebrate NAGS protein contains domains with a carbamate kinase fold and an acyl-CoA N-acyltransferase fold, and protein crystallization experiments are currently underway. Inherited NAGS deficiency results in hyperammonemia, presumably due to the loss of CPSI activity. We, and others, have recently identified mutations in families with neonatal and late-onset NAGS deficiency and the identification of the gene has now made carrier testing and prenatal diagnosis feasible. A structural analog of NAG, carbamylglutamate, has been shown to bind and activate CPSI, and several patients have been reported to respond favorably to this drug (Carbaglu).

Fig. 1

Alignment of vertebrate NAGS proteins. Legend: hNAGS, human; mNAGS, mouse; fNAGS, fugu; and zfNAGS, zebrafish. Missense mutations found in patients or those generated by random mutagenesis in the mouse sequence are marked in yellow, and the resulting amino acid change is labeled above the alignment. Mutations known to be deleterious are shown in red. Strongly conserved blocks are shaded in gray. Residues predicted to be active site residues are marked with dots below the alignment. Arrows at position 51 and 92 indicate the start sites of the two processed NAGS proteins dubbed “short mature” and “conserved domain.” Discrepancies between mutation numbering and alignment position number are due to insertions when generating the alignment.

Fig. 2

Hidden Markov model based alignment of human NAGS sequence with E. coli N-acetylglutamate kinase (NAGK) and with the histone acetyltransferase domain of P300/CBP associating factor (PCAF). Active site residues determined from the crystal structures of NAGK and PCAF are highlighted in red, along with their counterparts in the NAGS sequence. The alignment between hNAGS and NAGK has several shortcomings, including long insertions in the region of active site residues and relatively poor conservation of active site residues. This argues that the alignment cannot be used to correctly identify the NAG binding residues within NAGS.

Fig. 3

Liquid chromatography–mass spectrometry based NAGS enzyme assay. Left panel: typical stable isotope dilution sample. The ratio between a known amount of isotopic NAG and the NAG produced in the assay is used to quantitate NAG formation. Right panel: separation conditions can be modified to simultaneously monitor glutamate, carbamylglutamate, and NAG. Peaks corresponding to glutamate, N-acetylglutamate, and N-carbamylglutamate are shown with the chemical structures of N-carbamylglutamate and N-acetylglutamate.

Fig. 4

Identification of processing sites in NAGS. (Left) SDS–PAGE of nickel affinity purified NAGS expressed in insect cells. Three major isoforms are visible corresponding to preprotein (A), “short mature” (B), and “conserved domain” (C). (Right) MS spectra and MS/MS spectra following in gel tryptic digestion of the uppermost band. All three bands contain a characteristic 1150 m/z peak in their tryptic MS spectra that corresponds to the peptide LAFALAFLQR of NAGS upon analysis using MS/MS mode.