Expression pattern and biochemical properties of zebrafish N-acetylglutamate synthase

Abstract

The urea cycle converts ammonia, a waste product of protein catabolism, into urea. Because fish dispose ammonia directly into water, the role of the urea cycle in fish remains unknown. Six enzymes, N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase III, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase and arginase 1, and two membrane transporters, ornithine transporter and aralar, comprise the urea cycle. The genes for all six enzymes and both transporters are present in the zebrafish genome. NAGS (EC 2.3.1.1) catalyzes the formation of N-acetylglutamate from glutamate and acetyl coenzyme A and in zebrafish is partially inhibited by L-arginine. NAGS and other urea cycle genes are highly expressed during the first four days of zebrafish development. Sequence alignment of NAGS proteins from six fish species revealed three regions of sequence conservation: the mitochondrial targeting signal (MTS) at the N-terminus, followed by the variable and conserved segments. Removal of the MTS yields mature zebrafish NAGS (zfNAGS-M) while removal of the variable segment from zfNAGS-M results in conserved NAGS (zfNAGS-C). Both zfNAGS-M and zfNAGS-C are tetramers in the absence of L-arginine; addition of L-arginine decreased partition coefficients of both proteins. The zfNAGS-C unfolds over a broader temperature range and has higher specific activity than zfNAGS-M. In the presence of L-arginine the apparent Vmax of zfNAGS-M and zfNAGS-C decreased, their Km(app) for acetyl coenzyme A increased while the Km(app) for glutamate remained unchanged. The expression pattern of NAGS and other urea cycle genes in developing zebrafish suggests that they may have a role in citrulline and/or arginine biosynthesis during the first day of development and in ammonia detoxification thereafter. Biophysical and biochemical properties of zebrafish NAGS suggest that the variable segment may stabilize a tetrameric state of zfNAGS-M and that under physiological conditions zebrafish NAGS catalyzes formation of N-acetylglutamate at the maximal rate.

Figure 1. Relative expression of urea cycle genes in developing zebrafish.

mRNA levels were measured at nine developmental stages: 32 cells, 30% epiboly (4.6 hpf), 90% epiboly (9 hpf), tailbud (10 hpf), 24 hpf, 48 hpf, 72 hpf, 96 hpf, 105 hpf, and normalized to the abundance of each mRNA in adult zebrafish. The scales of y-axes differ due to different expression patterns of zebrafish urea cycle genes.

Figure 2. Sequence conservation and domain structure of fish NAGS proteins.

A. Domain structure of fish NAGS. MTS – mitochondrial targeting signal shown in blue; VS – variable segment shown in yellow; AAK – amino acid kinase domain shown in red; NAT – N-acetyltransferase domain shown in green. B. Sequence alignment of the N-terminal region of six fish NAGS proteins. Predicted MTS are shown in blue typeface. The variable segment is highlighted in yellow. The first 33–35 amino acids of the AAK domain are shown in red typeface.

Figure 3. Purification of recombinant zfNAGS-M and zfNAGS-C.

The zfNAGS-M (A) and zfNAGS-C (B) with the N-terminal polyhistidine tag were overexpressed in E. coli and purified using nickel-affinity column. Lane 1 – cell lysate; lane 2 – flow-through fraction; lane 3 – wash fraction; lane 4 – elution with 125 mM imidazole; lane 5 – elution with 250 mM imidazole; lane 6 – elution with 500 mM imidazole.

Figure 4. Effect of L-arginine on biochemical properties of zfNAGS-M and zfNAGS-C. K_m ^app and apparent V_max for AcCoA (A and C) and glutamate (B and D) when increasing amounts of arginine were added to zfNAGS-M and zfNAGS–C.

(Blue – V_max; Magenta – K_m ^app). Error bars represent standard errors of the fitting parameters for the Michaelis-Menten equation.

Figure 5. Analytical gel chromatography of zfNAGS-M with and without L-arginine.

The top panel shows a semi-logarithmic plot of molecular mass vs. elution volume. Open circles correspond to elution volumes of blue dextran (2000 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), and myoglobin (16 kDa). Lower panels show absorption at 280 nm as a function of elution volume. Concentration of zfNAGS-M loaded on the column is indicated in each panel. Dark blue – elution profiles of zfNAGS-M without arginine. Cyan – elution profiles of zfNAGS-M in the presence of 1 mM L-arginine.

Figure 6. Analytical gel chromatography of zfNAGS-C with and without L-arginine.

The top panel shows a semi-logarithmic plot of molecular mass vs. elution volume. Open circles correspond to elution volumes of blue dextran (2000 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), and myoglobin (16 kDa). Lower panels show absorption at 280 nm as a function of elution volume. The concentration of zfNAGS-C loaded on the column is indicated in each panel. Dark blue - elution profiles of zfNAGS-C without L-arginine. Cyan – elution profiles of zfNAGS-C in the presence of 1 mM L-arginine.

Figure 7. Thermofluor® analysis of zebrafish NAGS in the presence and absence of L- and D-arginine.

Unfolding of zfNAGS-M was measured in the presence of increasing concentrations of either L-arginine (A) or D-arginine (B). Unfolding of zfNAGS-C was measured in the presence of increasing concentrations of either L-arginine (C) or D-arginine (D). Dark blue – thermal unfolding in the absence of L- or D-arginine. Cyan - thermal unfolding in the presence of 1 mM L- or D-arginine. Orange - thermal unfolding in the presence of 10 mM L- or D-arginine.