Limits...
Functional characterization and expression analysis of rice δ(1)-pyrroline-5-carboxylate dehydrogenase provide new insight into the regulation of proline and arginine catabolism.

Forlani G, Bertazzini M, Zarattini M, Funck D - Front Plant Sci (2015)

Bottom Line: Cations were found to modulate enzyme activity, whereas anion effects were negligible.This implies that millimolar levels of arginine would increase the affinity of P5C dehydrogenase toward its specific substrate.Results are discussed in view of the involvement of the enzyme in either proline or arginine catabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Biotechnology, University of Ferrara Ferrara, Italy.

ABSTRACT
While intracellular proline accumulation in response to various stress conditions has been investigated in great detail, the biochemistry and physiological relevance of proline degradation in plants is much less understood. Moreover, the second and last step in proline catabolism, the oxidation of δ(1)-pyrroline-5-carboxylic acid (P5C) to glutamate, is shared with arginine catabolism. Little information is available to date concerning the regulatory mechanisms coordinating these two pathways. Expression of the gene coding for P5C dehydrogenase was analyzed in rice by real-time PCR either following the exogenous supply of amino acids of the glutamate family, or under hyperosmotic stress conditions. The rice enzyme was heterologously expressed in E. coli, and the affinity-purified protein was thoroughly characterized with respect to structural and functional properties. A tetrameric oligomerization state was observed in size exclusion chromatography, which suggests a structure of the plant enzyme different from that shown for the bacterial P5C dehydrogenases structurally characterized to date. Kinetic analysis accounted for a preferential use of NAD(+) as the electron acceptor. Cations were found to modulate enzyme activity, whereas anion effects were negligible. Several metal ions were inhibitory in the micromolar range. Interestingly, arginine also inhibited the enzyme at higher concentrations, with a mechanism of uncompetitive type with respect to P5C. This implies that millimolar levels of arginine would increase the affinity of P5C dehydrogenase toward its specific substrate. Results are discussed in view of the involvement of the enzyme in either proline or arginine catabolism.

No MeSH data available.


Effect of anions and cations on the activity of P5C dehydrogenase. The effects of the addition of increasing concentrations of monovalent cations (A), divalent cations (B), heavy metals (C), various anions (D), and non-ionic solutes (E) to the reaction mixture were assessed using NAD+ as the electron acceptor. Cations were added as chlorides, with the exception of Ca2+, Mg2+, Be2+, Cu2+, Zn2+, and Fe2+, which were added as sulfates. Anions were added as potassium salts. To minimize the carry-over of chloride anions, L-P5C concentration was reduced to 250 μM, and the P5C solution was neutralized just before use with 1 M NH3. Additionally, Hepes buffer was used at 12.5 mM to reduce the presence of potassium cations in the standard reaction mixture. In all cases, three replicates were carried out for each treatment. Data were expressed as percent of untreated controls, and mean values ± SE are presented.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4525382&req=5

Figure 10: Effect of anions and cations on the activity of P5C dehydrogenase. The effects of the addition of increasing concentrations of monovalent cations (A), divalent cations (B), heavy metals (C), various anions (D), and non-ionic solutes (E) to the reaction mixture were assessed using NAD+ as the electron acceptor. Cations were added as chlorides, with the exception of Ca2+, Mg2+, Be2+, Cu2+, Zn2+, and Fe2+, which were added as sulfates. Anions were added as potassium salts. To minimize the carry-over of chloride anions, L-P5C concentration was reduced to 250 μM, and the P5C solution was neutralized just before use with 1 M NH3. Additionally, Hepes buffer was used at 12.5 mM to reduce the presence of potassium cations in the standard reaction mixture. In all cases, three replicates were carried out for each treatment. Data were expressed as percent of untreated controls, and mean values ± SE are presented.

Mentions: Since P5C dehydrogenase from potato cultured cells had been found to be inhibited by NaCl concentrations above 100 mM (Forlani et al., 1997a), the possibility that high solute levels may reduce the activity of P5C dehydrogenase (and proline oxidation in turn) was considered. Because P5C neutralization and reaction mixture buffering may increase both anion and cation concentration in the assay environment, assay conditions were modified to limit the carry-over of ions. Under these conditions, the addition of non-ionic solutes up to 1 M to the reaction mixture was ineffective (Figure 10E). Anions exerted quite slight effects, and only bicarbonate and nitrate (but not chloride) were inhibitory at concentrations lower than 200 mM (Figure 10D). Interestingly, cations were much more potent inhibitors of P5C dehydrogenase. Li+ and Na+ progressively inhibited enzyme activity above 10 mM, whereas K+ and NH+4 were ineffective below 200 mM (Figure 10A). Divalent cations, also not considering the non-physiological Be2+ ions, were even more inhibitory, with a 32% reduction of the catalytic rate by Mg2+ at a concentration of 10 mM (Figure 10B). Very similar data were obtained when using NADP+ instead of NAD+ as the electron acceptor (not shown).


Functional characterization and expression analysis of rice δ(1)-pyrroline-5-carboxylate dehydrogenase provide new insight into the regulation of proline and arginine catabolism.

Forlani G, Bertazzini M, Zarattini M, Funck D - Front Plant Sci (2015)

Effect of anions and cations on the activity of P5C dehydrogenase. The effects of the addition of increasing concentrations of monovalent cations (A), divalent cations (B), heavy metals (C), various anions (D), and non-ionic solutes (E) to the reaction mixture were assessed using NAD+ as the electron acceptor. Cations were added as chlorides, with the exception of Ca2+, Mg2+, Be2+, Cu2+, Zn2+, and Fe2+, which were added as sulfates. Anions were added as potassium salts. To minimize the carry-over of chloride anions, L-P5C concentration was reduced to 250 μM, and the P5C solution was neutralized just before use with 1 M NH3. Additionally, Hepes buffer was used at 12.5 mM to reduce the presence of potassium cations in the standard reaction mixture. In all cases, three replicates were carried out for each treatment. Data were expressed as percent of untreated controls, and mean values ± SE are presented.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525382&req=5

Figure 10: Effect of anions and cations on the activity of P5C dehydrogenase. The effects of the addition of increasing concentrations of monovalent cations (A), divalent cations (B), heavy metals (C), various anions (D), and non-ionic solutes (E) to the reaction mixture were assessed using NAD+ as the electron acceptor. Cations were added as chlorides, with the exception of Ca2+, Mg2+, Be2+, Cu2+, Zn2+, and Fe2+, which were added as sulfates. Anions were added as potassium salts. To minimize the carry-over of chloride anions, L-P5C concentration was reduced to 250 μM, and the P5C solution was neutralized just before use with 1 M NH3. Additionally, Hepes buffer was used at 12.5 mM to reduce the presence of potassium cations in the standard reaction mixture. In all cases, three replicates were carried out for each treatment. Data were expressed as percent of untreated controls, and mean values ± SE are presented.
Mentions: Since P5C dehydrogenase from potato cultured cells had been found to be inhibited by NaCl concentrations above 100 mM (Forlani et al., 1997a), the possibility that high solute levels may reduce the activity of P5C dehydrogenase (and proline oxidation in turn) was considered. Because P5C neutralization and reaction mixture buffering may increase both anion and cation concentration in the assay environment, assay conditions were modified to limit the carry-over of ions. Under these conditions, the addition of non-ionic solutes up to 1 M to the reaction mixture was ineffective (Figure 10E). Anions exerted quite slight effects, and only bicarbonate and nitrate (but not chloride) were inhibitory at concentrations lower than 200 mM (Figure 10D). Interestingly, cations were much more potent inhibitors of P5C dehydrogenase. Li+ and Na+ progressively inhibited enzyme activity above 10 mM, whereas K+ and NH+4 were ineffective below 200 mM (Figure 10A). Divalent cations, also not considering the non-physiological Be2+ ions, were even more inhibitory, with a 32% reduction of the catalytic rate by Mg2+ at a concentration of 10 mM (Figure 10B). Very similar data were obtained when using NADP+ instead of NAD+ as the electron acceptor (not shown).

Bottom Line: Cations were found to modulate enzyme activity, whereas anion effects were negligible.This implies that millimolar levels of arginine would increase the affinity of P5C dehydrogenase toward its specific substrate.Results are discussed in view of the involvement of the enzyme in either proline or arginine catabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science and Biotechnology, University of Ferrara Ferrara, Italy.

ABSTRACT
While intracellular proline accumulation in response to various stress conditions has been investigated in great detail, the biochemistry and physiological relevance of proline degradation in plants is much less understood. Moreover, the second and last step in proline catabolism, the oxidation of δ(1)-pyrroline-5-carboxylic acid (P5C) to glutamate, is shared with arginine catabolism. Little information is available to date concerning the regulatory mechanisms coordinating these two pathways. Expression of the gene coding for P5C dehydrogenase was analyzed in rice by real-time PCR either following the exogenous supply of amino acids of the glutamate family, or under hyperosmotic stress conditions. The rice enzyme was heterologously expressed in E. coli, and the affinity-purified protein was thoroughly characterized with respect to structural and functional properties. A tetrameric oligomerization state was observed in size exclusion chromatography, which suggests a structure of the plant enzyme different from that shown for the bacterial P5C dehydrogenases structurally characterized to date. Kinetic analysis accounted for a preferential use of NAD(+) as the electron acceptor. Cations were found to modulate enzyme activity, whereas anion effects were negligible. Several metal ions were inhibitory in the micromolar range. Interestingly, arginine also inhibited the enzyme at higher concentrations, with a mechanism of uncompetitive type with respect to P5C. This implies that millimolar levels of arginine would increase the affinity of P5C dehydrogenase toward its specific substrate. Results are discussed in view of the involvement of the enzyme in either proline or arginine catabolism.

No MeSH data available.