Enzymes Involved in the Biosynthesis of Arginine from Ornithine in Maritime Pine (Pinus pinaster Ait.)

Abstract

The amino acids arginine and ornithine are the precursors of a wide range of nitrogenous compounds in all living organisms. The metabolic conversion of ornithine into arginine is catalyzed by the sequential activities of the enzymes ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASSY) and argininosuccinate lyase (ASL). Because of their roles in the urea cycle, these enzymes have been purified and extensively studied in a variety of animal models. However, the available information about their molecular characteristics, kinetic and regulatory properties is relatively limited in plants. In conifers, arginine plays a crucial role as a main constituent of N-rich storage proteins in seeds and serves as the main source of nitrogen for the germinating embryo. In this work, recombinant PpOTC, PpASSY and PpASL enzymes from maritime pine (Pinus pinaster Ait.) were produced in Escherichia coli to enable study of their molecular and kinetics properties. The results reported here provide a molecular basis for the regulation of arginine and ornithine metabolism at the enzymatic level, suggesting that the reaction catalyzed by OTC is a regulatory target in the homeostasis of ornithine pools that can be either used for the biosynthesis of arginine in plastids or other nitrogenous compounds in the cytosol.

Keywords: Pinus; amino acid biosynthesis; arginine; biochemical regulation; conifers; enzyme kinetics; nitrogen metabolism; ornithine.

Figure 1

Arginine biosynthesis. Schematic pathway of arginine biosynthesis from ornithine with the main acting molecules and possible metabolic destinations for arginine. * Although the implication of arginine in the biosynthesis of NO has been established in animals, it remains a matter of debate in plants. NAGS: N-acetylglutamate synthase; NAGK: N-acetylglutamate kinase; NAGPR: N-acetylglutamyl-phosphate reductase; NAOAT: N-acetylornithine aminotransferase; NAOGAcT: N-acetylornithine:glutamate acetyltransferase; NAOD: N-acetylornithine deacetylase; OTC: Ornithine transcarbamilase; ASSY: Argininosuccinate synthetase; ASL: Argininosuccinate lyase.

Figure 2

Schematic representation of OTC, ASSY and ASL primary structures. The distribution of conserved protein motifs and the primary structures of the proteins in both plants and animals are shown, with the conserved sequences and the punctual changes in some motifs (underlined). It is also highlighted the predicted cleavage site of the transit peptide using bioinformatics algorithms (TargetP 2.0). “Animal consensus” refers to the integration of sequences from Homo sapiens, Mus musculus, Rattus norvegicus, Anas platyrhynchos, Bos taurus, Ovis aries and Danio rerio. Complete alignment for these enzymes is shown in Figure S1.

Figure 3

Molecular mass estimations of PpOTC, PpASSY and PpASL. Samples from purified proteins were loaded separately onto a size exclusion ENrich™ SEC 650 column (Bio-Rad). Molecular masses were calculated by comparing the partition coefficient (Kav) of the samples with the standards used to calibrate the column: thyroglobulin (669 kDa), apoferritin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa) and carbonic anhydrase (29 kDa).

Figure 4

Sigmoidal kinetics of PpOTC. The graph shows the saturation curve of the enzyme in the presence of ornithine (a) and carbamoyl-P (b). A Hill plot of the kinetic data is shown in the inset (nH = 3.47 ± 0.38 for ornithine; nH = 0.48 ± 0.03 for carbamoyl-P).

Figure 5

Effect of different compounds on the enzyme activities of PpOTC, PpASSY and PpASL. (a) The histogram shows the enzymatic activity of the three enzymes in the presence of different amino acids, spermine (Spn), spermidine (Spd) and succinic acid (Succ). N.d. for no determined data. The enzyme assays were performed in the presence of different compounds at a final concentration of 10 mM as described in the Material and Methods section. Enzyme activity levels in the absence of compounds were considered the controls, C (100%), and the corresponding values were 2.3 ± 0.1 nkat/μg for PpOTC, 0.21 ± 0.02 nkat/μg for PpASSY and 15.63 ± 0.41 nkat/μg for ASL. (b) Inhibition plots showing PpOTC, PpASSY and PpASL activities in the presence of increasing amounts of selected effectors indicated in panel (a). (*) Significant difference vs. the control. All reactions were carried out with substrate concentrations greater than their respective K_m (mM) values at 25 °C with vigorous shaking during 5 min (end point).

Figure 6

Allosteric inhibition of PpOTC by arginine. The figure shows how the presence of arginine modifies the PpOTC activity, reducing V_max but increasing the affinity of the enzyme for ornithine. Values: 0 mM Arg: S_0.5 = 1.12 ± 0.23 mM, V_max = 0.57 ± 0.05 nkat/μg; 10 mM Arg: S_0.5 = 0.32 ± 0.08 mM, V_max = 0.37 ± 0.01 nkat/μg; 20 mM Arg: S_0.5 = 0.08 ± 0.06 mM, V_max = 0.25 ± 0.01 nkat/μg.