Effect of arginine on oligomerization and stability of N-acetylglutamate synthase

Abstract

N-acetylglutamate synthase (NAGS; E.C.2.3.1.1) catalyzes the formation of N-acetylglutamate (NAG) from acetyl coenzyme A and glutamate. In microorganisms and plants, NAG is the first intermediate of the L-arginine biosynthesis; in animals, NAG is an allosteric activator of carbamylphosphate synthetase I and III. In some bacteria bifunctional N-acetylglutamate synthase-kinase (NAGS-K) catalyzes the first two steps of L-arginine biosynthesis. L-arginine inhibits NAGS in bacteria, fungi, and plants and activates NAGS in mammals. L-arginine increased thermal stability of the NAGS-K from Maricaulis maris (MmNAGS-K) while it destabilized the NAGS-K from Xanthomonas campestris (XcNAGS-K). Analytical gel chromatography and ultracentrifugation indicated tetrameric structure of the MmMNAGS-K in the presence and absence of L-arginine and a tetramer-octamer equilibrium that shifted towards tetramers upon binding of L-arginine for the XcNAGS-K. Analytical gel chromatography of mouse NAGS (mNAGS) indicated either different oligomerization states that are in moderate to slow exchange with each other or deviation from the spherical shape of the mNAGS protein. The partition coefficient of the mNAGS increased in the presence of L-arginine suggesting smaller hydrodynamic radius due to change in either conformation or oligomerization. Different effects of L-arginine on oligomerization of NAGS may have implications for efforts to determine the three-dimensional structure of mammalian NAGS.

Figure 1. Thermofluor analysis of NgNAGS, MmNAGS-K and XcNAGS-K in the presence and absence of L-arginine and D-arginine.

Unfolding of bacterial NgNAGS was measured in the presence of increasing concentrations of either L-arginine (A) or D-arginine (B). Unfolding of MmNAGS-K was measured in the presence of increasing concentrations of either L-arginine (C) or D-arginine (D). Unfolding of XcNAGS-K was measured in the presence of increasing concentrations of either L-arginine (E) or D-arginine (F). Cyan and blue – thermal unfolding in the absence of L- or D-arginine. Magenta - 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.

Figure 2. Thermal inactivation of MmNAGS-K (A) and XcNAGS-K (B).

Specific synthase activity was normalized to specific activity measured after incubation at 30 °C. Blue – relative synthase activity in the absence of L-arginine. Orange – relative synthase activity in the presence of 1 mM L-arginine.

Figure 3. Thermofluor analysis of mouse NAGS in the presence and absence of L- and D-arginine.

Unfolding of mNAGS-M was measured in the presence of increasing concentrations of either L-arginine (A) or D-arginine (B). Unfolding of mNAGS-C was measured in the presence of increasing concentrations of either L-arginine (C) or D-arginine (D). Cyan and blue – thermal unfolding in the absence of L- or D-arginine. Magenta - 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.

Figure 4

Thermal inactivation of mNAGS-M (A) and mNAGS-C (B). Specific NAGS activity was normalized to specific activity measured after incubation at 30 °C. Blue – relative synthase activity in the absence of L-arginine after incubation without L-arginine. Orange – relative synthase activity in the presence of 1 mM L-arginine after incubation with 1 mM L-arginine. Magenta – fold-increase of specific NAGS activity in the presence of 1 mM L-arginine.

Figure 5

Concentration dependence of Stokes’ radii of MmNAGS-K (A), XcNAGS-K (B), mNAGS-M (C) and mNAGS-C (D). Stokes’ radii were determined using analytical gel chromatography (Supplementary Figs S2 and S3) in the presence (orange) and absence (blue) of L-arginine.

Figure 6. Analytical ultracentrifugation of the XcNAGS-K.

(A) Distribution of sedimentation coefficients c(s) in the absence (blue) and presence (orange) of L-arginine at two different protein concentrations. Molar concentrations refer to the concentration of XcNAGS-K monomer. (B) The S_w isotherm for XcNAGS-K in the absence of arginine showing the concentration dependence of the weight-average sedimentation coefficient in the c(s) distributions integrated between 7 and 16 S (solid circles) in overlay with the best-fit curve describing a tetramer-octamer association model.

Figure 7. Analytical gel chromatography of MmNAGS-K and XcNAGS-K.

(A) and (C) MmNAGS-K and XcNAGS-K that were allowed to equilibrate overnight at indicated protein concentrations with and without L-arginine. (B and D) MmNAGS-K and XcNAGS-K were diluted immediately before loading onto column and did not equilibrate with L-arginine prior to loading onto column. The top panels show a semi-logarithmic plot of molecular mass vs. elution volume of size and molecular weight standard proteins. Lower panels show absorption at 280 nm as a function of elution volume and protein concentration. Blue - elution profiles in the absence of L-arginine. Orange – elution profiles in the presence of 1 mM L-arginine.

Figure 8

Elution profiles of MmNAGS-K (A) and XcNAGS-K (B). Absorption at 280 nm (dotted lines) and rate of product formation (solid lines) were measured as functions of elution volume in the absence (blue) or presence (orange) of L-arginine.

Figure 9. Analytical gel chromatography of mNAGS-C and mNAGS-M.

(A and C) mNAGS-C and mNAGS-M that were allowed to equilibrate overnight at indicated protein concentrations with and without L-arginine. (B and D) mNAGS-C and mNAGS-M were diluted immediately before loading onto column and did not equilibrate with L-arginine prior to loading onto column. The top panels show a semi-logarithmic plot of molecular mass vs. elution volume of size and molecular weight standard proteins. Lower panels show absorption at 280 nm as a function of elution volume and protein concentration. Blue - elution profiles in the absence of L-arginine. Orange – elution profiles in the presence of 1 mM L-arginine.

Figure 10. Elution profiles of mNAGS-C and and mNAGS-M.

(A and C) mNAGS-C and mNAGS-M that were allowed to equilibrate overnight with and without L-arginine. (B and D) mNAGS-C and mNAGS-M did not equilibrate with L-arginine prior to loading onto column. Absorption at 280 nm (dotted lines) and rate of product formation (solid lines) were measured as functions of elution volume in the absence (blue) or presence (orange) of L-arginine.